Synthesis of coelenterazine

ABSTRACT

Disclosed herein are synthesis methods for coelenterazine. Also disclosed are articles including the coelenterazine and coelenterazine derivatives. Representative absorbent articles include disposable diapers and adult incontinence products.

This application is a continuation-in-part application of U.S.application Ser. No. 16/457,788, filed on Jun. 28, 2019, which claimsthe benefit of U.S. Provisional Application No. 62/845,189, filed May 8,2019, and U.S. Provisional Application No. 62/692,485, filed Jun. 29,2018; and which also claims the benefit of U.S. application Ser. No.16/457,732, filed Jun. 28, 2019, which claims the benefit the benefit ofU.S. Provisional Application No. 62/753,024, filed Oct. 30, 2018, andU.S. Provisional Application No. 62/692,502, filed Jun. 29, 2018; eachof which applications is expressly incorporated herein by reference inits entirety.

BACKGROUND

Personal care absorbent products, such as infant diapers, adultincontinent pads, and feminine care products, typically contain a fluidabsorbent core. Many absorbent articles include the fluid absorbent coredisposed between a top sheet and a back sheet. The top sheet can beformed from a fluid-permeable material adapted to promote fluid transferinto the absorbent core, such as upon a liquid insult, usually withminimal fluid retention by the top sheet. U.S. southern pine fluff pulpis commonly used in the absorbent core, generally in the form of afibrous matrix, and sometimes in conjunction with a superabsorbentpolymer (SAP) dispersed throughout the fibrous matrix. This fluff pulpis recognized worldwide as the preferred fiber for absorbent products,based on factors such as the fluff pulp's high fiber length, fibercoarseness, and its relative ease of processing from a wet-laid anddried pulp sheet to an air-laid web. The raw material for this type ofcellulosic fluff pulp is Southern Pine (e.g., Loblolly Pine, Pinus taedaL.). The raw material is renewable, and the pulp is easilybiodegradable. Compared to SAP, these fibers are inexpensive on a permass basis but tend to be more expensive on per unit of liquid heldbasis. These fluff pulp fibers mostly absorb within the intersticesbetween fibers. For this reason, a fibrous matrix readily releasesacquired liquid on application of pressure. The tendency to releaseacquired liquid can result in significant skin wetness during use of anabsorbent product that includes a core formed exclusively fromcellulosic fibers. Such products also tend to leak the acquired liquidbecause liquid is not effectively retained in such a fibrous absorbentcore.

SAPs are water-swellable, generally water-insoluble absorbent materialshaving a high absorbent capacity for fluids. They are used in absorbentarticles like baby diapers or adult incontinent products to absorb andhold body fluids. SAP, upon absorption of fluids, swells and becomes agel holding more than its weight of such fluids. The SAPs in common useare mostly derived from acrylic acid. Acrylic acid-based polymers alsocomprise a meaningful portion of the cost structure of diapers andincontinent pads. SAPs are designed to have high gel strength (asdemonstrated by high absorbency under load or AUL). The high gelstrength (upon swelling) of currently used SAP particles helps them toretain significant void space between particles, which is helpful forrapid fluid uptake. However, this high “void volume” simultaneouslyresults in significant interstitial (between particles) liquid in theproduct in the saturated state. When there is interstitial liquid the“rewet” value or “wet feeling” of an absorbent product is compromised.

Some absorbent articles, such as diapers or adult incontinence pads,also include an acquisition and distribution layer (ADL) for thecollection, and uniform and timely distribution of fluid from a fluidinsult to the absorbent core. An ADL is usually placed between the topsheet and the absorbent core, and normally takes the form of compositefabric with most likely the top-one third of the fabric having lowdensity (higher denier fiber) with relatively large voids and highervoid volume for the effective acquisition of the presented fluid, evenat relatively higher discharge rates. The middle one-third of thecomposite fabric of the ADL is usually made of higher density (lowdenier) fibers with smaller voids, while the lower one-third of thefabric is made of even higher density (lower and smaller denier) fibersand yet with finer voids. The higher density portions of the compositehave more and finer capillaries and hence develop greater capillarypressure, thus moving greater volumes of fluid to the outer regions ofthe structure thus enabling the proper channelization and distributionof the fluid in an evenly fashion to allow the absorbent core to take upall of the liquid insult in a time bound manner to allow SAP within theabsorbent core to hold and to gel the insult neither too slow nor toofast. The ADL provides for more rapid liquid acquisition (minimizingflooding in the target zone), and ensures more rapid transport andthorough distribution of the fluid into the absorbent core.

As noted above, the absorbent core is adapted to retain fluid, and assuch may consist of one or more layers, such as layers to acquire,distribute, and/or store fluid. In many cases, a matrix of cellulosefibers, such as in the form of an air-laid pad and/or non-woven web, isused in (or as) the absorbent core of absorbent articles. In some cases,the different layers may consist of one or more different types ofcellulose fibers, such as cross-linked cellulose fibers. The absorbentcore may also include one or more fluid retention agents, such as one ormore SAPs, distributed throughout the fiber matrix, usually asparticles. Advances in SAP technology have allowed the design ofabsorbent core configurations in which fluff pulp contributes less tothe absorbent capability of the core and more to providing a matrixstructure in which the SAP is stably held. Fluff pulp fibers alsoprovide fluid distribution functionality, to direct fluid to the SAP.However, it has been found that these structural and fluid distributionfunctions may be provided, in some configurations, by synthetic fibers,leading to the development of absorbent cores containing both fluff pulpfibers and synthetic fibers, and even “fluff-less” absorbent corescontaining no fluff pulp fibers. These configurations may offer theadvantage of being less physically bulky, without sacrificingabsorbency.

The back sheet is typically formed from a fluid-impermeable material toform a barrier to prevent retained fluid from escaping.

Whatever the structure, when the absorbent article is wet from one ormore liquid insults, the chances for the fluid coming in contact withthe skin increases profoundly, and if left unchanged for a long time canresult in diaper rash for infants or dermatitis problem in adults,thereby posing a skin wellness hazard. However, in general, the only wayto know whether the diaper or the incontinent pad is dry or wet is tophysically inspect it. During day time this may not pose a significantproblem because a caregiver can check the diapers or adult incontinentproducts as many times as desired. However, inspections during nighttime can be a discomfort to the baby as well as to the adult, disturbingtheir sleep. Moreover, frequent night time inspections, such as severaltimes in a single night, can disrupt the wearer's sleeping pattern,which poses health hazard to baby as well as the adult patient.

In addition, it is typical that an article of clothing, such as pants,pajamas, and/or undergarments, is worn over the diaper or absorbentarticle. Accordingly, even absorbent articles that incorporate differenttypes of wetness and/or moisture indicators pose difficulties in timelydiscovery of an insult.

As a result, there it typically a time lapse between the insult and itsdiscovery. If this time period is prolonged, then there exists thepossibility of developing diaper rash, skin irritation, and/or skinflaking. These conditions can be very painful for those affected. Thisis particularly true for babies and those adults in care-givingfacilities, and particularly true for night time insults, which can leadto longer periods prior to changing the absorbent article.

Previous moisture indicators incorporated into absorbent articles usecolor change as a visual indication of wetness detection. Inks thatappear, or disappear, based on contact with liquid are popularmechanisms for wetness detection. Fluorescence has also been used forwetness detection, such as by incorporating a compound that fluorescesin the presence of a liquid. The mechanisms for such indicatorsgenerally fall into three broad categories: (1) imprinting a moistureindicating pattern on one of the piles of the absorbent article; (2)discrete moisture-indicating strips or layers that are incorporatedbetween the layers of the absorbent article; and (3) a discrete (i.e.,not part of the absorbent article's construction) indicating strip thatis fastened to the interior of the absorbent article immediately priorto use.

Whatever the mechanism, these visual indicators are deficient inlow-light (e.g., night time) situations. Appearing or disappearing inksmust be directly visually detected, such that the caregiver can see theabsorbent product. In low-light situations, this may require both alight source (e.g., overhead light or flashlight), as well as theremoval of covering garments (e.g., pajamas or undergarments).Fluorescent indicators suffer similar issues, in that they require anexternal light source to excite the fluorescent compound. Suchexcitation is typically provided by exposing the indicator to UV light(which presents health concerns to the wearer and caregiver) and must bein direct optical communication with the fluorescent compound, whichthen requires removal of covering garments, blankets, etc. Therefore,the use of visual indicators previously used to detect wetness inabsorbent garments suffers many disadvantages in low-light situations,which greatly reduces the usefulness of their indication mechanisms.

There is a need for chemiluminescent materials that can generate lightupon exposure to moisture, where the chemiluminescent materials arereadily synthesized in large yields and good purity. The presentdisclosure seeks to fulfill these needs and provides further advantages.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In one aspect, the present disclosure features a method of makingcoelenterazine, including: coupling4-(5-amino-6-benzylpyrazin-2-yl)phenol (coelenteramine) with3-(4-(benzyloxy)phenyl)-2-oxoprop anal to provide8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one;and deprotecting the8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-oneto provide8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one(coelenterazine).

In another aspect, the present disclosure features a method of making3-(4-(benzyloxy)phenyl)-2-oxopropanal, including providing1-(benzyloxy)-4-(chloromethyl)benzene, and reacting the1-(benzyloxy)-4-(chloromethyl)benzene in two steps to provide3-(4-(benzyloxy)phenyl)-2-oxopropanal.

In another aspect, the present disclosure features a method of makingcoelenterazine, including:

(a) reacting 3-benzylpyrazin-2-amine (25) with N-bromosuccinimide toprovide 3-benzyl-5-bromopyrazin-2-amine (2);

(b) reacting the 3-benzyl-5-bromopyrazin-2-amine (2) in two sequentialsteps to provide 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7)(coelenteramine); and

(c) coupling the 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) withsilyl-protected 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one to providecoelenterazine, or a salt thereof.

In yet another aspect, the present disclosure features a method ofmaking coelenterazine, including:

coupling 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) with3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one (23)to provide coelenterazine (16), or a salt thereof;

wherein the3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one (23)is synthesized by

-   -   i. reacting 4-hydroxybenzaldehyde (8) with        tert-butyldimethylsilyl chloride to provide        4-((tert-butyldimethylsilyl)oxy)benzaldehyde (20a);    -   ii. reacting the 4-((tert-butyldimethylsilyl)oxy)benzaldehyde        with sodium borohydride to provide        (4-((tert-butyldimethylsilyl)oxy)phenyl)methanol (20b);    -   iii. reacting the        (4-((tert-butyldimethylsilyl)oxy)phenyl)methanol with        methanesulfonyl chloride to provide        tert-butyl(4-(chloromethyl)phenoxy)dimethylsilane (21);    -   iv. reacting the        tert-butyl(4-(chloromethyl)phenoxy)dimethylsilane with magnesium        to provide (4-((tert-butyldimethylsilyl)oxy)benzyl)magnesium        chloride (22); and    -   v. reacting the        (4-((tert-butyldimethylsilyl)oxy)benzyl)magnesium chloride with        ethyl 2,2-diethoxyacetate to provide the        3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one        (23).

In yet another aspect, the present disclosure features a method ofmaking coelenterazine, including:

coupling 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14) to providecoelenterazine, or a salt thereof.

In yet a further aspect, the present disclosure features a method ofmaking coelenterazine, including:

(a) reacting 3,5-dibromopyrazin-2-amine and (bromomethyl)benzene in thepresence of zinc, iodine, and a first palladium catalyst to provide3-benzyl-5-bromopyrazin-2-amine (2);

(b) reacting 3-benzyl-5-bromopyrazin-2-amine (2) in a first step toprovide a hydrochloride salt of3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), and reacting thehydrochloride salt of 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) ina second step to provide 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7); and

(c) coupling 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14) to providecoelenterazine (16), or a salt thereof.

In yet another aspect, the present disclosure features a method ofmaking coelenterazine, including:

coupling 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14) for a duration of 16hours to 28 hours, to provide coelenterazine, or a salt thereof;

wherein the coupling reaction is monitored by reverse phase HPLC and isquenched when 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) stopsdepleting, or when coelenterazine starts to decompose.

In yet another aspect, the present disclosure features a method ofmaking coelenterazine, including:

reacting 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14) for a duration of 16hours to 28 hours to provide crude coelenterazine, or a salt thereof;

triturating crude coelenterazine with ethyl acetate; and

isolating a coelenterazine, or salt thereof.

In yet another aspect, the present disclosure features a method ofmaking coelenterazine, including (a) reacting pyrazin-2-amine (24) withbenzyl chloride to provide 3-benzylpyrazin-2-amine (25) (e.g., underGrignard conditions, such as by first providing a solution of magnesium,iodine, and ethyl bromide in a solvent before reacting pyrazin-2-amine(24) with benzyl chloride to provide 3-benzylpyrazin-2-amine (25); (b)reacting 3-benzylpyrazin-2-amine (25) with N-bromosuccinimide to provide3-benzyl-5-bromopyrazin-2-amine (2); (c) reacting the3-benzyl-5-bromopyrazin-2-amine (2) in two sequential steps to provide4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine); and (d)coupling the 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) withsilyl-protected 1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one orsilyl-protected 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one to providecoelenterazine (16), or a salt thereof.

In another aspect, the present disclosure features a method of making anabsorbent article, including incorporating the coelenterazine madeaccording to the methods of the present disclosure into an absorbentarticle.

In yet another aspect, the present disclosure features an absorbentarticle, including the coelenterazine synthesized by the methods of thepresent disclosure.

DETAILED DESCRIPTION

Disclosed herein are synthesis methods for making coelenterazine, inhigh yield and with good purity. Also disclosed are articles includingthe coelenterazine and coelenterazine derivatives. Representativeabsorbent articles include disposable diapers and adult incontinenceproducts.

Chemiluminescence results from a chemical reaction that produces lightand therefore provides a lighted indication of moisture that can be seenin low light and/or in the absence of light, and through clothes.Furthermore, chemiluminescence requires no external excitation light, asis required for photoluminescent (e.g., fluorescence) indicators.Accordingly, by generating light upon contact with an aqueous system(e.g., urine), the disclosed embodiments greatly enhance the ability ofabsorbent articles to indicate the occurrence of an insult in darkenedconditions (e.g., at night). Moreover, by generating light that can beseen through clothing, a caregiver may be able to ascertain theoccurrence of an insult without having to move or disturb the infant oradult wearer of such an absorbent article, such as during sleep.

The articles provided herein may provide the distinct advantages ofinsult indication at night and through clothes, which may reduce or eveneliminate the need for caregivers to disturb the sleep (e.g., by pullingdown clothes and/or shining a light) of one wearing an absorbent articlein order to test for an insult. Further, because light (e.g., visiblelight) is produced by the chemiluminescent systems disclosed herein,there is no need to expose the absorbent article and/or the wearer to UVlight in order to determine whether an insult has occurred, allowinghealth concerns associated with UV radiation to be avoided. Examples ofarticles including chemiluminescent materials are described, forexample, in U.S. application Ser. No. 14/516,255, the disclosure ofwhich is herein incorporated in its entirety.

The articles of the present disclosure provide improved ease with whichan insult can be detected, which allows the caregiver to check for aninsult more frequently, due to the reduced interruption required. Morefrequent checks may allow an insult to be detected sooner and theabsorbent article changed soon after the insult, thereby reducing theamount of time the insult contacts the wearer's skin, as well asreducing the possibility of fluid from multiple insults contacting thewearer's skin. The skin health and general comfort of the wearer areimproved when the length of time that fluid is in contact with the skinis reduced. In some embodiments, the component amounts/ratios can becalibrated so that instead of seeing a peak and then a fade after eachof a sequence of insults, once there is sufficient water present in theabsorbent article, the luminescence can be maintained at a relativelysteady intensity (e.g., the luminescence can vary less than about 30%,less than about 20%, less than about 10%, less than 30%, less than 20%,or less than 10%,) over a period of time (e.g., about 24 hours, about 12hours, about 6 hours, about 3 hours, about 2 hours, about 1 hour, about30 minutes, about 15 minutes, 24 hours, 12 hours, 6 hours, 3 hours, 2hours, 1 hour, 30 minutes, or 15 minutes). As used herein, the word“about” as it relates to a quantity indicates a number within range ofminor variation above or below the stated reference number. For example,“about” can refer to a number within a range of 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or 1% above or below the indicated reference number. In someembodiments, “about” refers to a number within a range of 5% above orbelow the indicated reference number. In some embodiments, “about”refers to a number within a range of 10% above or below the indicatedreference number. In some embodiments, “about” refers to a number withina range of 1% above or below the indicated reference number.

Synthesis of Coelenterazine

In one aspect, the present disclosure features a method of makingcoelenterazine, including coupling coelenteramine, or a salt thereof,with a protected 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one; orcoupling coelenteramine, or a salt thereof, with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14), to providecoelenterazine, or a salt thereof.

In another aspect, the present disclosure features a method of makingcoelenterazine, including coupling coelenteramine, or a salt thereof,with a protected 1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one (e.g., asilyl-protected 1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one), toprovide coelenterazine, or a salt thereof.

In another aspect, the present disclosure features a method of makingcoelenterazine, including coupling4-(5-amino-6-benzylpyrazin-2-yl)phenol (coelenteramine) with3-(4-(benzyloxy)phenyl)-2-oxoprop anal to provide8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one;and deprotecting the8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-oneto provide8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one(coelenterazine).

The coelenteramine can be made via different routes, as outlined below.The methods can provide coelenterazine in good yield and at good purity.

Coelenteramine Synthesis

In some embodiments, the coelenteramine is made by first (a1) reacting3-benzylpyrazin-2-amine (25) with N-bromosuccinimide to provide3-benzyl-5-bromopyrazin-2-amine (2), or a salt thereof or by (a2)reacting 3,5-dibromopyrazin-2-amine and (bromomethyl)benzene in thepresence of zinc, iodine, and a palladium catalyst to provide the3-benzyl-5-bromopyrazin-2-amine (2), or a salt thereof. In a step (b),the 3-benzyl-5-bromopyrazin-2-amine (2) is then reacted in twosequential steps to provide 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7)(coelenteramine).

The reaction of (a1) 3-benzylpyrazin-2-amine (25) withN-bromosuccinimide can be carried out in an organic solvent, such asCHCl₃ (chloroform) at room temperature (e.g., about 22° C. to 23° C.,22° C. to 23° C., or 22° C.) at atmospheric pressure (i.e., about 1 atm,or 1 atm). Once the reaction is complete, the reaction mixture can bewashed with water, and 3-benzyl-5-bromopyrazin-2-amine (2) can beisolated by removing the organic solvent under reduced pressure. In someembodiments, (a1) provides 3-benzyl-5-bromopyrazin-2-amine (2) in ayield of about 60% to about 85% (e.g., 60% to 85%) relative to3-benzylpyrazin-2-amine (25) and in a purity of at least about 85%(e.g., a purity of about 85% to about 95%, a purity of about 85% toabout 100%, a purity of 85% to 95%, or a purity of 85% to 100%). As usedherein, it is understood that % yield refers to mole % yield. As usedherein, the purity of a given compound (when accompanied by the % yieldof the given compound) refers to the weight percent purity relative tothe mass of the pure compound calculated based on mole % yield.

In some embodiments, in (a2), the palladium catalyst isbis(triphenylphosphine) palladium(II) dichloride. The palladium catalystcan be present in an amount of about 5 to about 10 mole percent (e.g., 5to 10 mole percent) relative to 3,5-dibromopyrazin-2-amine. In someembodiments, (a2) includes about 1:2 to about 1:3 molar equivalent(e.g., 1:2 to 1:3 molar equivalent) of 3,5-dibromopyrazin-2-amine to(bromomethyl)benzene. (a2) can provide 3-benzyl-5-bromopyrazin-2-amine(2) in a yield of about 55% to about 75% (e.g., 55% to 75%) relative tothe 3,5-dibromopyrazin-2-amine at a purity of about 80% to about 95%(80% to 95%). In some embodiments, (a2) includes reacting3,5-dibromopyrazin-2-amine and (bromomethyl)benzene for a duration ofabout 18 hours to about 30 hours (e.g., 18 hours to 30 hours). Thereaction of 3,5-dibromopyrazin-2-amine and the (bromomethyl)benzene canoccur at a temperature of about 25 to about 40° C. (e.g., 25 to 40° C.)at a pressure of about 1 atm (e.g., 1 atm).

In some embodiments, step (b) includes a first step of reacting the3-benzyl-5-bromopyrazin-2-amine (2) with 4-methoxyphenyl boronic acid (4L) in the presence of a palladium catalyst to provide3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), or salt thereof. Thepalladium catalyst can be a palladium (0) catalyst, for example,tetrakis(triphenylphosphine)palladium(0). The palladium catalyst can bepresent in an amount of about 5 to about 10 percent by weight (e.g., 5to 10 percent by weight) relative to the 3-benzyl-5-bromopyrazin-2-amine(2). The first step in (b) can include about 1:1 to about 1:1.3 molarequivalent of the 3-benzyl-5-bromopyrazin-2-amine (2) to 4-methoxyphenylboronic acid (4 L). In some embodiments, the3-benzyl-5-bromopyrazin-2-amine (2) and the 4-methoxyphenyl boronic acid(4 L) are reacted together for a duration of about 120 minutes to about300 minutes (e.g., 120 minutes to 300 minutes). The3-benzyl-5-bromopyrazin-2-amine (2) and the 4-methoxyphenyl boronic acid(4 L) can be reacted together at a temperature of about 60° C. to about90° C. (e.g., 60° C. to 90° C.) and at atmospheric pressure (i.e., apressure of about 1 atm, or 1 atm). The first step in (b) can providethe 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), or salt thereof, ina yield of about 60% to about 85% (e.g., 60% to 85%) relative to the3-benzyl-5-bromopyrazin-2-amine (2) of the product at a purity of about80 to about 95% (i.e., a yield of 60% to 85% of the 80%-95% pureproduct). As used here, yield at a given purity range refers to theyield of the product having the described purity range.

Alternatively, in some embodiments, (b) includes a first step ofreacting the 3-benzyl-5-bromopyrazin-2-amine (2) with(4-methoxyphenyl)boronic acid in the presence of a palladium catalyst(e.g., a palladium (II) catalyst, such as bis(benzonitrile)palladium(II) dichloride) to provide3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) or a salt thereof. Thepalladium (II) catalyst can be present in an amount of about 5 to about10 (e.g., 5 to 10) mole percent relative to the3-benzyl-5-bromopyrazin-2-amine (2). The reaction mixture can furtherinclude 1,4-bis(diphenylphosphino)butane, for example, in an amount ofabout 5 to about 10 (e.g., 5 to 10) mole percent relative to the3-benzyl-5-bromopyrazin-2-amine (2). In some embodiments, the reactionmixture further includes toluene, aqueous sodium carbonate, and ethanol.The 3-benzyl-5-bromopyrazin-2-amine (2) can be reacted with the(4-methoxyphenyl)boronic acid for a duration of about 200 minutes toabout 350 minutes (e.g., 200 to 350 minutes), and/or at a temperature ofabout 80 to about 110° C. (e.g., 80 to 100° C.) and at a pressure ofabout 1 atm (e.g., 1 atm). When the palladium (II) catalyst is used,3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) can be provided in ayield of about 65% to about 85% (e.g., 65% to 85%) relative to the3-benzyl-5-bromopyrazin-2-amine (2). The3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) can be isolated bydiluting the reaction mixture with water and extracting with ethylacetate. The ethyl acetate extract can be removed from the extract underreduced pressure to provide 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine(5).

The 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), or salt thereof,regardless of whether obtained with a palladium (0) or palladium (II)catalyst, can be isolated by precipitation of the3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) as a hydrochloride salt.

Precipitation is advantageous as it can eliminate column chromatography,which can be laborious, costly, and time-consuming; and/or can alsoincrease yield relative to column chromatography. In some embodiments,isolating the 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) does notinclude chromatography (e.g., liquid chromatography), does not includerecrystallization, or does not include chromatography andrecrystallization. In some embodiments, the reaction mixture from thereaction of 3-benzyl-5-bromopyrazin-2-amine (2) with(4-methoxyphenyl)boronic acid is diluted with aqueous sodium chloridesolution (e.g., an about 20% (e.g., 20%) by weight sodium chloridesolution) and extracted with ethyl acetate. The ethyl acetate extractcan then be treated with HCl aqueous solution (e.g., an about 3N (e.g.,3N) HCl_((aq)) solution) and the3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine hydrochloride salt product(5) can be isolated by filtration with a purity of about 75% to about95% (e.g., 75% to 95%).

In some embodiments, (b) further includes a second step of deprotectingthe 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), or salt thereof, toprovide the 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine).The deprotection can include subjecting the3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) to pyridinium chloride.Treatment with pyridinium chloride can proceed at an elevatedtemperature of about 180 to about 220° C. (e.g., 180 to 220° C., or 200°C.) at atmospheric pressure. In some embodiments, the elevatedtemperature can cause the pyridinium chloride to separate from thereaction mixture by evaporation from the reaction mixture.Alternatively, in certain embodiments, deprotection can includesubjecting the 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) to sodiumhydride and ethanethiol in N,N′-dimethyl-formamide (DMF). When treatedwith sodium hydride and ethanethiol in DMF, the reaction mixture can beat a temperature of about 90° C. to about 120° C. (e.g., about 100° C.to 110° C., 90° C. to 120° C., or 100° C. to 110° C.) and/or for aperiod of about 15 minutes to about 5 hours (e.g., about 30 minutes toabout 2 hours, about 30 minutes to about 1 hour, 30 minutes to 2 hours,30 minutes to 1 hour, or 30 minutes). Once the reaction is complete, themixture can be cooled to about 30° C. to about 50° C. (e.g., about 35°C. to about 45° C., about 40° C., 35° C. to 45° C., or 40° C.),extracted with water and an organic solvent (e.g., ethyl acetate). Theorganic layer can then be separated, refluxed, and then cooled to about5° C. to 20° C. (e.g., about 10° C. to 15° C., 5° C. to 20° C., or 10°C. to 15° C.), the 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7)(coelenteramine) can be isolated by filtration.

In both deprotection methods, the coelenteramine can be optionallypurified by washing with an aqueous sodium hydroxide/dioxane solution,stirring with activated charcoal/silica, filtration, followed byprecipitation by acidification of the filtrate, and isolation of theprecipitated product by filtration. The deprotection can provide4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine) in a yieldof about 90% to about 100% (e.g., 90% to 100%, at least about 95%, atleast 95%, at least about 98%, at least 98%, at least about 99%, or atleast 99%) at a purity of about 85% to about 100% (e.g., 85% to 100%,about 90%, or 90%) relative to3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5).

In some embodiments, coelenteramine is synthesized by (a) reacting3,5-dibromopyrazin-2-amine and (bromomethyl)benzene in the presence ofzinc, iodine, and a first palladium catalyst to provide3-benzyl-5-bromopyrazin-2-amine (2); (b) reacting3-benzyl-5-bromopyrazin-2-amine (2) with 4-methoxyphenyl boronic acid (4L) in the presence of a palladium catalyst in a first step to provide ahydrochloride salt of 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5),and deprotecting the hydrochloride salt of3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) in a second step toprovide 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine).

In some embodiments, coelenteramine is synthesized by reactingpyrazin-2-amine with benzyl chloride to provide 3-benzylpyrazin-2-amine;reacting 3-benzylpyrazin-2-amine (25) with N-bromosuccinimide to provide3-benzyl-5-bromopyrazin-2-amine (2); and reacting the3-benzyl-5-bromopyrazin-2-amine (2) in two sequential steps to provide4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine). The twosequential steps for providing coelenteramine from3-benzyl-5-bromopyrazin-2-amine (2) can include a first step of reactingthe 3-benzyl-5-bromopyrazin-2-amine (2) with silyl-protected4-bromophenol in the presence of magnesium and a palladium catalyst toprovide silyl-protected 4-(5-amino-6-benzylpyrazin-2-yl)phenol. In someembodiments, the palladium catalyst istetrakis(triphenylphosphine)palladium(0), which can be present in anamount of 1 percent or more (e.g., 2 percent or more, 3 percent or more,4 percent or more, 5 percent or more, 6 percent or more, 7 percent ormore, 8 percent or more, or 9 percent or more) and/or 10 percent or less(e.g., 9 percent or less, 8 percent or less, 7 percent or less, 6percent or less, 5 percent or less, 4 percent or less, 3 percent orless, or 2 percent or less) by weight relative to the3-benzyl-5-bromopyrazin-2-amine (2). For example, for every 100 grams of3-benzyl-5-bromopyrazin-2-amine (2), 1 to 10 grams (e.g., 1 gram, 2grams, 3 grams, or 5 grams) of palladium catalyst can be used. As anexample, for every 100 grams of 3-benzyl-5-bromopyrazin-2-amine (2), 1gram of palladium catalyst can be used. The two sequential steps caninclude a second step of deprotecting the silyl-protected4-(5-amino-6-benzylpyrazin-2-yl)phenol to provide the4-(5-amino-6-benzylpyrazin-2-yl)phenol (7), for example by subjectingthe silyl-protected 4-(5-amino-6-benzylpyrazin-2-yl)phenol to aqueousHCl. This synthesis procedure reduces or eliminates the use of: n-butyllithium-in the first step of the synthesis, by replacing the n-butyllithium reaction in toluene with benzyl chloride and THF(tetrahydrofuran). Thus, this synthesis presents the ability to scale upthe reaction chemistry and can reduce the cost of the synthesis. Inaddition, the changes can improve the overall safety of the chemistry byreplacing highly reactive materials with more stable materials. Theomission of boronic acid compounds in the synthesis can greatly reducethe amount of expensive palladium catalyst in the reaction.

In some embodiments, coelenteramine is synthesized by reactingpyrazin-2-amine (24) with benzyl chloride to provide3-benzylpyrazin-2-amine (25) (e.g., under Grignard conditions, such asby first providing a solution of magnesium, iodine, and ethyl bromide ina solvent before reacting pyrazin-2-amine (24) with benzyl chloride toprovide 3-benzylpyrazin-2-amine (25); reacting 3-benzylpyrazin-2-amine(25) with N-bromosuccinimide to provide 3-benzyl-5-bromopyrazin-2-amine(2); reacting 3-benzyl-5-bromopyrazin-2-amine (2) with 4-methoxyphenylboronic acid (4 L) in the presence of a palladium catalyst in a firststep to provide a hydrochloride salt of3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), and deprotecting thehydrochloride salt of 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) ina second step to provide 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7)(coelenteramine). In some embodiments, the deprotection of3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) includes exposing the3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) to an acidicenvironment, such as HBr in acetic acid, to provide4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine).

The reaction of 3-benzylpyrazin-2-amine (25) with N-bromosuccinimide canbe carried out in an organic solvent, such as CHCl₃ (chloroform) at roomtemperature (e.g., about 22° C. to 23° C., 22° C. to 23° C., or 22° C.)at atmospheric pressure (i.e., about 1 atm, or 1 atm). Once the reactionis complete, the reaction mixture can be washed with water, and/or anaqueous acid (e.g., HCl_((aq)), and 3-benzyl-5-bromopyrazin-2-amine (2)can be isolated by removing the organic solvent under reduced pressure.In some embodiments, 3-benzyl-5-bromopyrazin-2-amine (2) is provided ina yield of about 60% to about 85% (e.g., 70-75%) relative to3-benzylpyrazin-2-amine (25) and in a purity of at least about 85%(e.g., a purity of about 85% to about 95%, a purity of about 85% toabout 100%, a purity of 85% to 95%, a purity of 90-95%, or a purity of85% to 100%).

In some embodiments, the 3-benzyl-5-bromopyrazin-2-amine (2) is reactedwith 4-methoxyphenyl boronic acid (4 L) in the presence of a palladiumcatalyst to provide 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), orsalt thereof. The palladium catalyst can be a palladium (0) catalyst,for example, tetrakis(triphenylphosphine)palladium(0), or a palladium(II) catalyst, such as bis(benzonitrile) palladium(II) dichloride. Whena palladium (II) catalyst is used, the reaction mixture can furtherinclude 1,4-bis(diphenylphosphino)butane, for example, in an amount ofabout 5 to about 10 (e.g., 5 to 10) mole percent relative to the3-benzyl-5-bromopyrazin-2-amine (2). The palladium catalyst can bepresent in an amount of about 5 to about 10 percent by weight (e.g., 5to 10 percent by weight) relative to the 3-benzyl-5-bromopyrazin-2-amine(2). The first step in (b) can include about 1:1 to about 1:1.3 molarequivalent of the 3-benzyl-5-bromopyrazin-2-amine (2) to 4-methoxyphenylboronic acid (4 L). In some embodiments, the3-benzyl-5-bromopyrazin-2-amine (2) and the 4-methoxyphenyl boronic acid(4 L) are reacted together for a duration of about 120 minutes to about300 minutes (e.g., 120 minutes to 300 minutes). The reaction solvent caninclude 1,4-dioxane and/or water. In some embodiments, the reactionmixture further includes potassium carbonate in an amount of about 75 toabout 85 mole percent relative to the 3-benzyl-5-bromopyrazin-2-amine(2). The 3-benzyl-5-bromopyrazin-2-amine (2) and the 4-methoxyphenylboronic acid (4 L) can be reacted together for a duration of about 12 toabout 36 hours (e.g., about 20 to 24 hours, about 12 to about 24 hours,or about 15 to 24 hours), and/or at a temperature of about 80 to about110° C. (e.g., 80 to 100° C., or 80 to 85° C.), and/or at atmosphericpressure (i.e., a pressure of about 1 atm, or 1 atm). The3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), or salt thereof, can beprovided in a yield of about 65% to about 95% (e.g., 85% to 90%, 80% to95%, or 85% to 90%) relative to the 3-benzyl-5-bromopyrazin-2-amine (2),at a purity of for example, 90% to 95% (e.g., 92% to 95%). The3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) can be isolated bydiluting the reaction mixture with water and extracting with ethylacetate. The ethyl acetate extract can be removed from the extract underreduced pressure to provide 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine(5).

The 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), or salt thereof,regardless of whether obtained with a palladium (0) or palladium (II)catalyst, can be isolated by precipitation of the3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) as a hydrochloride salt.Precipitation is advantageous as it can eliminate column chromatography,which can be laborious, costly, and time-consuming; and/or can alsoincrease yield relative to column chromatography. In some embodiments,isolating the 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) does notinclude chromatography (e.g., liquid chromatography), does not includerecrystallization, or does not include chromatography andrecrystallization. In some embodiments, the reaction mixture from thereaction of 3-benzyl-5-bromopyrazin-2-amine (2) with(4-methoxyphenyl)boronic acid is diluted with aqueous sodium chloridesolution (e.g., an about 20% (e.g., 20%) by weight sodium chloridesolution) and extracted with ethyl acetate. The ethyl acetate extractcan then be treated with HCl aqueous solution (e.g., an about 3N (e.g.,3N) HCl_((aq)) solution) and the3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine hydrochloride salt product(5) can be isolated by filtration with a purity of about 75% to about95% (e.g., 75% to 95%).

The 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), or salt thereof,can then be deprotected to provide4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine). In someembodiments, the deprotection includes exposing the3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5), or salt thereof, to anacid (e.g., HBr and acetic acid). For example, the HBr can have an HBrconcentration of 48% in water, and the aqueous HBr can be mixed withacetic acid in a ratio of from about 1:2 (e.g., from about 1:1.5, fromabout 1:1.25) to about 1:1 (e.g., to about 1:1.25, or to about 1:1.5).The deprotection can occur at a temperature of from about 100° C. (e.g.,from about 105° C., from about 110° C., from about 115° C.) to about120° C. (e.g., to about 115° C., to about 110° C., to about 105° C.) fora duration of from about 5 hours (e.g., from about 8 hours, from about10 hours, from about 12 hours, or from about 14 hours) to about 18 hours(e.g., to about 14 hours, to about 12 hours, to about 10 hours, or to 8hours), for example, from 8 to 10 hours, at atmospheric pressure. Afterthe deprotection reaction, the reaction mixture can be cooled, extractedwith an organic solvent (such as ethyl acetate), and the organic solventcan be removed under reduced pressure. The residue can then be refluxedwith a hydrocarbon (such as cyclohexane), filtered, isolated, and driedto provide 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine).The 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine) can beobtained in an yield of from about 70% (e.g., from about 75%, from about80%, or from 85%) to about 90% (e.g., to about 85%, to about 80%, or toabout 75%), for example, from 75% to 80%, at a purity of from about 85%(e.g., from about 87%, from about 90%, or from about 92%) to about 95%(e.g., to about 92%, (e.g., to about 90%, or to about 87%), for example,a purity of about 90%, relative to3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5).

Protected 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one synthesis

In some embodiments, the silyl protected1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one is3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one(23). The3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one (23)can be synthesized by: i) reacting 4-hydroxybenzaldehyde (8) withtert-butyldimethylsilyl chloride to provide4-((tert-butyldimethylsilyl)oxy)benzaldehyde (20a); ii) reacting the4-((tert-butyldimethylsilyl)oxy)benzaldehyde with sodium borohydride toprovide (4-((tert-butyldimethylsilyl)oxy)phenyl)methanol (20b); iii)reacting the (4-((tert-butyldimethylsilyl)oxy)phenyl)methanol withmethanesulfonyl chloride in the presence of a base (e.g., triethylamine)to provide tert-butyl(4-(chloromethyl)phenoxy)dimethylsilane (21); iv)reacting the tert-butyl(4-(chloromethyl)phenoxy)dimethylsilane withmagnesium, to provide (4-((tert-butyldimethylsilyl)oxy)benzyl)magnesiumchloride (22); and v) reacting the(4-((tert-butyldimethylsilyl)oxy)benzyl)magnesium chloride with ethyl2,2-diethoxyacetate to provide the3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one(23).

In some embodiments, the tert-butyldimethylsilyl protected1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one can degrade in acidicconditions, and can be stabilized in a basic environment. Thus, a basecan be added to the reaction mixture and/or during purification. Forexample, the tert-butyl(4-(chloromethyl)phenoxy)dimethylsilane (21) canbe purified by chromatography with an eluant that includestriethylamine. In some embodiments, the3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one (23)is further purified by chromatography with an eluant comprisingtriethylamine.

The 3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one(23) can be provided in a yield of about 20% to about 40% (e.g., 20% to40%, about 20% to 25%, or 20% to 25%) relative totert-butyl(4-(chloromethyl)phenoxy)dimethylsilane (21), at a purity ofabout 85% to about 95% (e.g., 85% to 95%, about 90%, or 90%).

1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14)

1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14) can be made, forexample, according to the reaction scheme below.

Synthesis of3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-dimethoxypropan-2-one)

In some embodiments, 4-(tert-butyldimethylsiloxy) benzyl chloride can beas synthesized above, and can be reacted in a Grignard reaction withmethyl 2,2-dimethoxyacetate to provide3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-dimethoxypropan-2-one).For example, the 4-(tert-butyldimethylsiloxy) benzyl chloride can bereacted with methyl 2,2-dimethoxyacetate in a Grignard reaction with Mg,and I₂ and dibromoethane as Grignard initiators to provide3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-dimethoxypropan-2-one).

Synthesis of 3-(4-(benzyloxy)phenyl)-2-oxopropanal

In some embodiments, the present disclosure features a method of making3-(4-(benzyloxy)phenyl)-2-oxopropanal, including providing1-(benzyloxy)-4-(chloromethyl)benzene, and reacting the1-(benzyloxy)-4-(chloromethyl)benzene in two steps to provide3-(4-(benzyloxy)phenyl)-2-oxopropanal. The method of making3-(4-(benzyloxy)phenyl)-2-oxopropanal does not include more than onepalladium-catalyzed reaction, from an initial starting material of4-hydroxybenzaldehyde.

The method can include a first step of reacting the1-(benzyloxy)-4-(chloromethyl)benzene with methyl 2,2-dimethoxyacetateunder Grignard conditions (e.g., ethyl bromide, magnesium, and acatalytic amount of iodine) to provide3-(4-(benzyloxy)phenyl)-1,1-dimethoxypropan-2-one. The3-(4-(benzyloxy)phenyl)-1,1-dimethoxypropan-2-one can be purified bysilica column chromatography.

The method can include a second step of reacting the3-(4-(benzyloxy)phenyl)-1,1-dimethoxypropan-2-one with acid (e.g.,aqueous acid, such as 10% aqueous HCl)) to provide the3-(4-(benzyloxy)phenyl)-2-oxopropanal. The3-(4-(benzyloxy)phenyl)-2-oxopropanal can be isolated in a yield of 60to 75% (e.g., 65% to 70%) at a purity of 85 to 95% (e.g., 90%) relativeto 1-(benzyloxy)-4-(chloromethyl)benzene.

In some embodiments, the intermediate1-(benzyloxy)-4-(chloromethyl)benzene can be made, for example, by thefollowing procedure. A mixture of 4-hydroxybenzaldehyde (8), benzylchloride, and anhydrous potassium carbonate in N,N-dimethyl formamidecan be formed, heated to a temperature of about 40-80° C. for a durationof from, for example, 5 hours to 3 days, under atmospheric pressure.After reaction completion, the mixture can be cooled to roomtemperature, charged with water, and centrifuged or filtered to isolatethe resulting 4-(benzyloxy)benzaldehyde.

The 4-(benzyloxy)benzaldehyde can then be reduced with sodiumborohydride to provide (4-(benzyloxy)phenyl)methanol. Briefly, thesodium borohydride can be added at a temperature of about 45 to 50° C.dropwise to a solution of 4-(benzyloxy)benzaldehyde in methanol. Thereaction mixture can then be cooled (e.g., to about 15° C.), acidified(e.g., with acetic acid), stirred with water, and the product can beisolated by filtration. The resultant product can be heated with anorganic solvent (e.g., n-hexane), filtered, and dried to provide(4-(benzyloxy)phenyl)methanol.

The (4-(benzyloxy)phenyl)methanol can then be reacted with thionylchloride to provide 1-(benzyloxy)-4-(chloromethyl)benzene (11). Forexample, a mixture of (4-(benzyloxy)phenyl)methanol in dichloromethaneand N,N′-dimethylformamide can be formed, to which thionyl chloride canbe added slowly at a temperature of from about 30 to about 35° C. Afterstirring the reaction for a duration of from about 30 minutes to about 2hours, the solvent can be removed, and the residue can be extracted withwater and an organic solvent, such as ethyl acetate. After separatingthe organic layer, the pH of the layer can be adjusted to about 8 to 9with an aqueous base, such as a soda ash aqueous solution, then theorganic layer can be separated again, washed with a sodium chlorideaqueous solution, and the organic layer is then separated andconcentrated under reduced pressure. The residue can then be washed withan organic solvent, such as n-hexane, and the product can be isolated byfiltration and dried to obtain the intermediate1-(benzyloxy)-4-(chloromethyl)benzene.

Coelenterazine

In some embodiments, the coelenterazine is obtained by coupling the4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) with silyl-protected1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one or silyl-protected1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one to provide coelenterazine,or a salt thereof. Alternatively, in some embodiments, thecoelenterazine is obtained by coupling4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14) to providecoelenterazine, or a salt thereof. The coupling step, whether withsilyl-protected 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one,silyl-protected 1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one, or with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14), can be conducted inthe presence of dioxane, water, and HCl (e.g., at a ratio of about90:5:5 dioxane to water to HCl; or at an HCl at 35%-38% concentration(i.e., concentrated HCl) to water ratio about 10: about 1 (e.g., 10:1)to about 1: about 1 (e.g., 1:1), where the dioxane:(HCl+H₂O) ratio isabout 9:1); and/or can be conducted at a temperature of about 60 toabout 90° C. (e.g., 60 to 90° C.) and at a pressure of about 1 atm(e.g., 1 atm); and/or can proceed for a duration of about 16 hours toabout 28 hours (e.g., 16 to 28 hours).

In some embodiments, whether the 4-(5-amino-6-benzylpyrazin-2-yl)phenol(7) is coupled with silyl-protected1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one, silyl-protected1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one, or with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14), the coupling reactioncan be conducted in a solvent mixture of dioxane, methanol, andisopropyl alcohol. The methanol and the isopropyl alcohol can eachindependently be in the solvent mixture at a concentration of 3% or more(e.g., 5% or more, 7% or more, or 9% or more and/or 10% or less (e.g.,9% or less, 7% or less, or 5% or less) by volume. In certainembodiments, the methanol and the isopropyl alcohol are eachindependently in the solvent mixture at a concentration of about 5%(e.g., 5%) by volume. In some embodiments, the4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) is reacted in the solventmixture of dioxane, methanol, and isopropyl alcohol with silyl-protected1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one, such as3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one(23). Without wishing to be bound by theory, it is believed thatmethanol can favor the reaction kinetics and yield by increasing thesolubility of the 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) in thesolvent mixture, thereby increasing its availability for reaction withsilyl-protected 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one,silyl-protected 1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one, or with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14). In some embodiments,the coupling reaction can be conducted at a temperature of about 60 toabout 90° C. (e.g., about 70° C. to 85° C., about 80° C., or 80° C.) andat a pressure of about 1 atm (e.g., 1 atm); and/or can proceed for aduration of about 24 hours to about 36 hours (e.g., 24 to 36 hours, or36 hours).

In some embodiments, the 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) iscoupled with silyl-protected1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (e.g.,3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one(23)), silyl-protected 1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one, orwith 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14) at a molar ratioof about 1:1.3 to about 1:2. In certain embodiments, the4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) is coupled withsilyl-protected 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (e.g.,3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one(23)) at a molar ratio of about 1:1.3 to about 1.2 (e.g., about 1:1.3,or 1:1.3).

In certain embodiments, whether the4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) is coupled withsilyl-protected 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one,silyl-protected 1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one, or with1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14), the coupling reactionis monitored by reverse phase HPLC and stopped when4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) stops depleting, or whencoelenterazine starts to decompose. Without wishing to be bound bytheory, it is believed that the reverse phase HPLC monitoring can beimportant, as the coelenterazine can start to decompose or degrade afterthe coelenteramine stops depleting.

In some embodiments, stopping the reaction mixture includes cooling thereaction mixture (e.g., to room temperature, about 23° C., or 23° C.),and optionally stirring the reaction mixture with activated carbon andsilica. The coelenterazine or a salt thereof can then be isolated byfiltering the reaction mixture (if activated carbon and silica areused), by removal of solvents (e.g., under reduced pressure),trituration with ethyl acetate, followed by filtration to obtain thesolid coelenterazine. In some embodiments, the coelenterazine isisolated as a salt, such as a hydrochloride salt.

In some embodiments, when silyl-protected1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one or silyl-protected1,1-dimethoxy-3-(4-hydroxyphenyl)propan-2-one is used as one of thestarting materials in the coupling reaction, the coelenterazine or asalt thereof is obtained in a yield of about 50% to about 70% (e.g., 50%to 70%) relative to the 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7), at apurity of about 55% to about 70% (e.g. 55% to 70%).

In some embodiments, when 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one(14) is used as one of the starting materials in the coupling reaction,coelenterazine or a salt thereof is obtained in a yield of about 60% toabout 70% (e.g., 60% to 70%) relative to the4-(5-amino-6-benzylpyrazin-2-yl)phenol (7), at a purity of about 60% toabout 75% (e.g., 60% to 75%). In some embodiments, the coelenterazine(or salt thereof) is obtained at a purity of 60% to 65% in the isolatedcomposition. Without wishing to be bound by theory, it is believed thatthe isolated coelenterazine, or salt thereof, is stabilized (e.g.,protected from degradation) by the presence of an amount of4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine). Thus, theisolated composition can include coelenterazine (or salt thereof) at anamount of 60% to 65% by weight and4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine). In someembodiments, the isolated composition consists essentially ofcoelenterazine (or salt thereof) at an amount of 60% to 65% by weightand 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine); suchthat impurities present in the composition do not substantiallycontribute (e.g., contribute more than 10%, more than 5%, or more than1%) to the luminescence of the coelenterazine, or salt thereof.

The relative amount of coelenterazine (or salt thereof) to4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine) in theisolated composition can be assessed by liquid chromatography-massspectrometry (LC-MS). For example, a 1 mg/ml methanolic solution of theisolated coelenterazine composition can be diluted ten times into aninjection solvent consisting of 70:30 reagent water:acetonitrile (v/v),each supplemented with 0.05% formic acid. The diluted solution includingthe isolated coelenterazine composition is separated by LC on a C-18reverse phase column using a gradient elution. In some embodiments, theseparation provides a response for coelenterazine (or salt thereof) atabout 1.7 min and a response for coelenteramine (7) at about 2.5 min.Tandem MS is configured to monitor the (M+H)+ parent ion of eachcompound which is subsequently fragmented into its characteristicdaughter ion. The daughter ion intensity creates the chromatographicsignal for each compound which is then integrated to produce an area forthe signal. The parent ion for coelenterazine is 424.1 Da with adaughter ion at 302.2 Da. The parent ion for coelenteramine is 278.1 Daand its daughter is 132.0 Da. In some embodiments, the ratio of thecoelenterazine (or salt thereof) to coelenteramine in the isolatedcomposition is about 20:1 or more (e.g., about 24:1 or more, about 30:1or more, about 40:1 or more, about 50:1 or more, about 60:1 or more,about 70:1 or more, about 80:1 or more, or about 90:1 or more) and/orabout 100:1 or less (e.g., about 90:1 or less, about 80:1 or less, about70:1 or less, about 60:1 or less, about 50:1 or less, about 40:1 orless, about 30:1 or less, or about 24:1 or less). In certainembodiments, the ratio of the coelenterazine (or salt thereof) tocoelenteramine in the isolated composition is 24:1 or more and/or 80:1or less. The isolated composition can be incorporated into an article,such as an absorbent article, as will be described below.

In some embodiments, the coelenterazine is obtained by coupling4-(5-amino-6-benzylpyrazin-2-yl)phenol (coelenteramine) with3-(4-(benzyloxy)phenyl)-2-oxopropanal to provide8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one;and deprotecting the8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-oneto provide8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one(coelenterazine).

Coupling the 4-(5-amino-6-benzylpyrazin-2-yl)phenol (coelenteramine)with the 3-(4-(benzyloxy)phenyl)-2-oxopropanal can occur in a solventmixture that includes dioxane, water, and HCl (e.g., concentrated HCl,36% HCl). The coupling reaction can be conducted at a temperature of 75°C. to 90° C. (e.g., 80° C. to 85° C.) for 12 to 36 hours (e.g., 24hours) in an inert atmosphere.

In some embodiments, deprotecting the8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-oneincludes a first deprotection step of exposing the8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-oneto acid, such as HCl (e.g., concentrated HCl, 36% HCl). The reactionmixture can include an organic solvent, such as dioxane. The acid can bepresent in an amount equal to or in excess of the volume of the solventmixture in the preceding coupling step. An intermediate deprotectedproduct can be obtained, for example, by filtering the reaction mixture,collecting the solid residue, and drying the solid residue beforeproceeding to the following step. The first deprotection step can beconducted at a temperature of 25° C. to 40° C. (e.g., 30 to 35° C.), fora duration of from 30 minutes (e.g., from 1 hour) to 2 hours (e.g., to1.5 hours). The intermediate deprotected product (i.e., the solidresidue) can be washed with an organic solvent (e.g., toluene) beforefiltering and drying.

Deprotecting the8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-onecan include a second deprotection step of exposing the driedintermediate deprotected product to acid, such as HCl (e.g.,concentrated HCl, 36% HCl). The reaction mixture can include an organicsolvent, such as dioxane. The second deprotection step can includeheating the intermediate deprotected product in HCl and the organicsolvent to a first temperature of about 50° C. to 75° C. (e.g., from 60°C. to 70° C., from 55° C. to 65° C., from 60° C. to 62° C.) for aduration of 6 to 24 hours, then at a second higher temperature (e.g.,higher than the first temperature by 5° C. to 15° C., by 5° C. to 10°C., by 10° C. to 15° C.) to provide coelenterazine. To isolate thecoelenterazine, the HCl and organic solvent can be removed under reducedpressure to provide a residue, the residue can be washed with organicsolvents such as ethyl acetate, then dichloromethane, and hexane. Theresidue can be dried under reduced pressure, at room temperature or upto a temperature of about 50° C. (e.g., 40-45° C.) to provide the finalcoelenterazine. The coelenterazine can be obtained in a yield of 70% ormore (e.g., 70% to 95%, 70% to 85%, 80% to 95%, 80% to 85%, or 80%) at apurity of from 55% to 70% (e.g., 55% to 65%, or 60% to 65%) relative to4-(5-amino-6-benzylpyrazin-2-yl)phenol.

The 3-(4-(benzyloxy)phenyl)-2-oxopropanal and the coelenteramine used inthe coupling reaction to provide coelenterazine can be made by themethods describe above.

Representative Syntheses

In some embodiments, the coelenterazine of the present disclosure can bemade according to Schemes A, B, and C, below. Scheme A illustrates thesynthesis of coelenteramine (Intermediate I), Scheme B illustrates thesynthesis of silyl-protected1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (Intermediate II), andScheme C illustrates the coupling of coelenteramine and silyl-protected1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one to generate coelenterazine.

In some embodiments, the coelenterazine of the present disclosure can bemade according to Schemes D, E, and F, below. Scheme D illustrates thesynthesis of coelenteramine, Scheme E illustrates the synthesis of1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14), and Scheme Fillustrates the coupling of coelenteramine and1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14) to generatecoelenterazine (16).

In some embodiments, the coelenterazine of the present disclosure can bemade according to Schemes G, H, and I, below. Scheme G illustrates thesynthesis of coelenteramine, Scheme H illustrates the synthesis of3-(4-(benzyloxy)phenyl)-2-oxopropanal, and Scheme F illustrates thecoupling of coelenteramine and 3-(4-(benzyloxy)phenyl)-2-oxopropanal,and subsequent deprotection of the intermediate product8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one,to generate8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one, coelenterazine (16).

Absorbent Articles

The coelenterazine, or salt thereof, made according to the methods ofthe present disclosure can be incorporated into an absorbent article.

In some embodiments, the present disclosure features an absorbentarticle, including the coelenterazine, or salt thereof, synthesized bythe methods of the present disclosure.

Materials and structural elements for absorbent articles that includeone or more components of a chemiluminescent system are described, forexample, in U.S. Provisional Application No. 62/692,502, titled“Chemiluminescent Wetness Indicator for Absorbent Products” and filed onJun. 29, 2018; U.S. Provisional Application No. 62/753,024, titled“Chemiluminescent Wetness Indicator for Absorbent Products” and filed onOct. 30, 2018; and U.S. application Ser. No. 16/457,732, titled“Chemiluminescent Wetness Indicator for Absorbent Products” and filed onJun. 28, 2019; the entire content of each of which is incorporatedherein by reference.

Chemilumines cent System

The chemiluminescent system is configured to produce light upon contactwith an aqueous system. The aqueous system initiates thechemiluminescence reaction in order to produce light. As used herein,the term “aqueous system” refers to water or water-containingcompositions. In the context of this disclosure, such water-containingcompositions are generally in the form of body fluid, such as urine,menses, fecal matter, and so forth. The occurrence of the release ofbodily fluid (or the fluid itself) is referred to herein as an “insult,”or “liquid insult” or “fluid insult.” Accordingly, the chemiluminescentsystems of the present disclosure produce light upon insult of anarticle in which the system is incorporated.

In being configured to produce light upon contact with an aqueoussystem, the chemiluminescent system includes at least one compound ormaterial that luminesces when contacted with an aqueous system. In oneembodiment, water initiates the chemiluminescence.

In one embodiment, the chemiluminescent system includes two or morematerials that luminesce when contacted with an aqueous system. In thisembodiment, there are two or more materials that together do notluminesce without the presence of the aqueous system.

Representative chemiluminescent systems that include two or morematerials include bioluminescent systems, such as a system that includesa luciferin and a luciferase.

Bioluminescence is light that is produced by a chemical reaction thatoccurs within the body or in the secretions of certain type oforganisms. Bioluminescence involves the combination of two types ofsubstances in a light-producing reaction: a luciferin and a luciferase.Luciferin is the compound that actually produces the light, for example,the luciferin can be coelenterazine. Luciferase is an enzyme thatcatalyzes the reaction. In some cases the luciferase is a protein knownas a photoprotein, and the light making process requires a charged ion(e.g., a cation such as calcium) to activate the reaction. Photoproteinis a variant of luciferase in which factors required for light emission(including luciferin and oxygen) are bound together as one unit. Often,the bioluminescence process requires the presence of a substance such asoxygen or adenosine triphosphate (ATP) to initiate the oxidationreaction. The reaction rate for the luciferin is controlled by theluciferase or photoprotein. The luciferin-luciferase reaction can alsocreate byproducts such as inactive oxyluciferin and water.

Luciferin and luciferase are generic names rather than specificmaterials. For example, the luciferin coelenterazine (natural form) iscommon in marine bioluminescence but variants can be chemicallysynthesized, and these various forms are collectively called luciferins.In another example, dinoflagellates (marine planktons) that obtain foodthrough photosynthesis use a luciferin that resembles the chlorophyllstructure.

The mechanism of light production through a chemical reactiondifferentiates bioluminescence from other optical phenomenon such asfluorescence or phosphorescence.

For example, fluorescent molecules do not emit their own light. Theyneed an external photon source to excite the electrons to a higherenergy state. On relaxation from the high energy state to their naturalground state, they release their acquired energy as a light source, butusually at a longer wavelength. Since the excitation and relaxationoccurs simultaneously, fluorescent light is seen only when illuminated(excited).

The term phosphorescence technically refers to a special case ofoptically excited light emission where the relaxation from the excitedstate to ground state, unlike the fluorescence, is not immediate, andthe photon emission persists for seconds to minutes after the originalexcitation.

The technical distinction between bioluminescence and fluorescence issometimes blurred in a practical context but technically they are twodistinct phenomena. In most cases, a bioluminescent can be anautofluorescent but the reverse is not true for a fluorescent; thelatter still requires photon for excitation to emit light. In some casesa bioluminescent cnidarians or crustaceans or fish can contain afluorescent protein like Green Fluorescent Protein (GFP) and the lightemitted from the bioluminescent would act as photons to excite the GFP.The GFP in turn under relaxed state would emit a light of different wavelength (most probably of higher wave length) than the wavelength of thebioluminescent light that it has received as photon. In this example,the GFP may be excited by a blue light emitted by the bioluminescent(wavelength about 470 nm, or 470 nm) but in turn would emit a greenlight under its relaxed state (wavelength of about 510 nm to about 520nm, or 510 nm to 520 nm).

Bioluminescent systems can be incorporated into fluff pulp compositions,fiber matrices, or absorbent articles in any manner that produces thedesired chemiluminescence.

In one embodiment, the fluff pulp composition or absorbent productcomprises a luciferin selected from the group consisting ofcoelenterazine, dinoflagellate luciferin, bacterial luciferin, fungalluciferin, firefly luciferin, and vargulin. With regard tocoelenterazine, there are many variants, any of which can be used in thefluff pulp composition.

Certain embodiments of coelenterazine consistent with this disclosurecomprise one or more of native coelenterazine, methyl coelenterazine,coelenterazine 400a (2-2′(4-dehydroxy)) coelenterazine, coelenterazinee, coelenterazine f, coelenterazine h, coelenterazine coelenterazine n,coelenterazine cp, coelenterazine ip, coelenterazine fcp, andcoelenterazine hep. As a further example, the coelenterazine may be oneor more of native coelenterazine, coelenterazine 400a, methylcoelenterazine, coelenterazine f, coelenterazine cp, coelenterazine fcp,and coelenterazine hep. As yet a further example, the coelenterazine maybe one or more of coelenterazine 400a, methyl coelenterazine andcoelenterazine fcp. As yet a further example, the coelenterazine may beone or more of coelenterazine 400a, methyl coelenterazine, andcoelenterazine hep. In yet another example, the coelenterazine may bemay be one or more of coelenterazine 400a and coelenterazine hep.

In one embodiment, the luciferin has a concentration of 0.0005% to0.002%, by weight of the fluff pulp. In one embodiment, the luciferinhas a concentration of 0.0005% to 0.0015%, by weight of the fluff pulp.In one embodiment, the luciferin has a concentration of 0.0005% to0.001%, by weight of the fluff pulp.

In some embodiments, the luciferin can be incorporated in any componentof an absorbent article. For example, the luciferin (e.g.,coelenterazine, or a coelenterazine salt of the present disclosure) canbe incorporated into an absorbent article in an amount of from about0.01 to about 100 mg (e.g., from about 0.01 to about 75 mg, from about0.01 to about 50 mg, from about 0.01 to about 25 mg, from about 0.01 toabout 10 mg, or from about 0.01 to about 5 mg), or 0.01 to 100 mg (e.g.,from 0.01 to 75 mg, from 0.01 to 50 mg, from 0.01 to 25 mg, from 0.01 to10 mg, or from 0.01 to 5 mg).

In one embodiment, the fluff pulp composition or absorbent productcomprises luciferase selected from the group consisting of Gaussialuciferase (Gluc), Renilla luciferase (RLuc), dinoflagellate luciferase,firefly luciferase, fungal luciferase, bacterial luciferase, and vargulaluciferase. Certain embodiments of the luciferase consistent with thisdisclosure comprise one or more of Gaussia luciferase, Renillaluciferase, dinoflagellate luciferase, and firefly luciferase. As afurther example, the luciferase may be one or more of Gaussialuciferase, Renilla luciferase, dinoflagellate luciferase, and fireflyluciferase. In yet a further example, the luciferase may be one or moreof Gaussia luciferase and Renilla luciferase.

In one embodiment, the luciferase has a concentration of about 0.005% toabout 0.04% (e.g., 0.005% to 0.04%) by weight of the fluff pulp. In oneembodiment, the luciferase has a concentration of about 0.005% to about0.02% (e.g., 0.005% to 0.02%) by weight of the fluff pulp. In oneembodiment, the luciferase has a concentration of about 0.005% to about0.01% (e.g., 0.005% to 0.01%) by weight of the fluff pulp.

In some embodiments, the luciferase can be incorporated in any componentof an absorbent article. For example, the luciferase (e.g., GLuc) can beincorporated into an absorbent article in an amount of from about 0.2 mgto about 40 mg (e.g., from about 0.2 mg to about 30 mg, from about 0.2mg to about 20 mg, from about 0.2 mg to about 15 mg, from about 0.2 mgto about 10 mg, from about 0.2 mg to about 5 mg, or from about 0.2 toabout 2 mg); or from 0.2 mg to 40 mg (e.g., from 0.2 mg to 30 mg, from0.2 mg to 20 mg, from 0.2 mg to 15 mg, from 0.2 mg to 10 mg, from 0.2 mgto 5 mg, or from 0.2 to 2 mg).

In one embodiment, the chemiluminescent system comprises coelenterazineas the luciferin and Gaussia or Renilla luciferase.

Representative luciferins include those of the coelenterazine family.Coelenterazine in its native form as well as its analogs have differentluminescent characteristics due to variation in their structuralmoieties. Given structural variations within the coelenterazine family,some are good substrates for certain luciferases, whereas some are not.Below is a brief description of native coelenterazine and representativeanalogs.

Coelenterazine (native form) is a luminescent enzyme substrate forRenilla (reniformis) luciferase (Rluc). Renillaluciferase/coelenterazine has also been used as the bioluminescencedonor in bioluminescence resonance transfer (BRET) studies.

Coelenterazine 400a is a derivative of coelenterazine and is a goodsubstrate for Renilla luciferase, but does not oxidize well with Gaussialuciferase (Gluc). It is the preferred substrate for BRET(bioluminescence resonance energy transfer) because its emission maximumof about 400 nm (e.g., 400 nm) has minimal interference with the GFPemission.

Fluorescence resonance energy transfer (FRET), BRET, resonance energytransfer (RET), and electronic energy transfer (EET) are mechanismsdescribing energy transfer between two light-sensitive molecules(chromophores) and can define the interference of a luminescent chemicalwith another luminescent chemical's energy transfer. A donorchromophore, initially in its electronic excited state, may transferenergy to an acceptor chromophore through nonradiative dipole-dipolecoupling. The efficiency of this energy transfer is inverselyproportional to the sixth power of the distance between donor andacceptor, making FRET extremely sensitive to small changes in distance.Measurements of FRET

efficiency can be used to determine if two fluorophores are within acertain distance of each other.

Such measurements are used as a research tool in fields includingbiology and chemistry.

Coelenterazine cp in a coelenterazine-aequorin complex generatesluminescence intensity about 15 times (e.g., 15 times) higher thancoelenterazine (native form).

Coelenterazine f has about 20 times (e.g., 20 times) higher luminescenceintensity (coelenterazine-apoaequorin complex) than the native formcoelenterazine, while its emission maximum is about 8 nm (e.g., 8 nm)longer than that of the native form.

Coelenterazine fcp is an analog wherein the a-benzene structure in thecoelenterazine moiety of coelenterazine f structure is replaced with acyclic pentane (similar to coelenterazine cp). Coelenterazine fcp hasluminescence intensity about 135 times (e.g., 135 times) greater thanthat of coelenterazine (native form).

Coelenterazine fcp complexes with aequorin to form a coelenterazinefcp-apoaequorin complex, and, as a substrate for aequorin, has arelative luminescence intensity of about 135 times (e.g., 135 times)that of native coelenterazine. However, coelenterazine fcp is a poorsubstrate for Renilla luciferase.

Other representative analogs of coelenterazine, as a substrate forRenilla Luciferase enzyme, are coelenterazine e, h and n. While thesethree analogs are good to excellent substrates for Renilla luciferase,they are poor substrates for apoaequorin.

The luminescent properties of coelenterazine analogs vary. For example,certain analogs emit less light (as measured as lumens) but with higherluminescent intensity (lumens/steradian). Table A lists the luminescentproperties of coelenterazine (native form) and its analogs with RenillaLuciferase. Luminescent intensity is reported as a % initial intensity.For example, an analog having an initial intensity of 900% is about 20times (e.g., 20 times) intense as compared to the native coelenterazinewith an initial intensity of about 45% (e.g., 45%).

TABLE A Luminescent Properties of Selected Coelenterazine Analogs withRenilla Luciferase Analog Aem(nm) Total Light(%} Initial Intensity(%)native 475 100 45 e 418, 475 137 900 f 473 28 45 h 475 41 135 n 475 47900 cp 470 23 135

Light is produced by the chemiluminescent system. The light is visuallydetectable by a caregiver in the dark and through clothing, and as suchthe light has a wavelength, intensity, and duration sufficient toprovide the necessary indication. These spectral characteristics of thechemiluminescent system can be tailored based on the chemiluminescentcompound or compounds. For example, in bioluminescent systems, theluciferin and luciferase can be selected to produce the desired lightcharacteristics. Depending on the bioluminescent system used, differentspectral characteristics can occur. In the presence of superoxide anionsand/or peroxynitrile compounds, coelenterazine can also emit lightindependent of enzyme (luciferase) oxidation, a process known asautoluminescence.

The chemiluminescent system can be tailored to produce particular colorsof light. As noted above in Table A, even within the coelenterazinefamily, the emission wavelength can range from about 400 nm (violet,e.g., 400 nm) to about 475 nm (blue with green tint, e.g., 475 nm).

With regard to duration, the duration of the light emitted may becontrolled by the selection of the coelenterazine (luciferin), in nativeform versus its analogues, and the enzyme (Luciferase), for exampleGaussia versus Renilla. The ratio and the concentration of luciferin andluciferase used may also modify the duration of light emission. To givean illustrative and non-limiting example, the luciferin analogue,coelenterazine e, has a total light of 130% and initial intensity of900% over native coelenterazine. By judiciously selecting theconcentration of coelenterazine e and Renilla luciferase, the durationof the light emitted can last as long as about 8 to about 10 hours(e.g., 8 to 10 hours).

In one embodiment, the light has a duration of about 0.5 to about 6hours (e.g., 0.5 to 6 hours). In another embodiment, the light has aduration of about 1 to about 4 hours (e.g., 1 to 4 hours). In anotherembodiment, the light has a duration of about 2 to about 3 hours (e.g.,2 to 3 hours).

With regard to intensity, quantum efficiency of the chemiluminescencecontributes to the intensity, depth, and hue of the color of theemission.

Quantum efficiency (QE) is the fraction of photon flux used to excite aluminescence chemical to elevate it to higher energy state. Quantumefficiency is one of the most important parameters used to evaluate thequality of a detector and is often called the “spectral response” toreflect its wavelength dependence. It is defined as the number of signalelectrons created per incident photon. In some cases it can exceed 100%(i.e. when more than one electron is created per incident photon). Ifthe spectral response exceeds 100%, then the intensity and depth of thecolor emitted is vivid, but depending on the status of the excited stateof the primary electron, the duration of the emission will be determined(i.e., the higher the excited state, the more time it takes to return tothe ground (normal) state).

Spectral responsivity is a similar measurement, but it has differentunits; the metric being the amperes or watts (i.e., how much currentcomes out of the device per incoming photon of a given energy andwavelength).

Both the quantum efficiency and the spectral responsivity are functionsof the photons' wavelength. For example, in the case of the luciferincoelenterazine, between the native form and one of its analogs,coelenterazine e, the latter has not only high light intensity but emits30% more light energy than the former, because the latter uponexcitation by a given quanta (hv) of incident photon generates twoelectrons and the primary electron at wavelength 475 has the sameemission intensity as native coelenterazine but with lumen intensityabout 20 times (e.g., 20 times) greater than that of the native product.Accordingly, the light emitted by the excited coelenterazine analogwould be twenty times brighter than the native form but with a totallight energy of about 130% (e.g., 130%) will last longer than the nativeform.

The wavelength determines the color of the emitted light.

In one embodiment, the fluff pulp composition includes a luciferin and aluciferase. Such a fluff pulp has both elements of the chemiluminescentsystem required to luminesce upon contact with an aqueous system.However, in another embodiment, the fluff pulp composition includes atleast one component selected from a luciferin and a luciferase. In suchan embodiment, the fluff pulp composition may include only one of aluciferin and a luciferase. Such a fluff pulp composition may beincorporated into an absorbent article such that the other one of theluciferin and the luciferase may be disposed in a top sheet or otherlayer of the absorbent article, such that the two components arecombined only when carried by a liquid insult (e.g., water from anaqueous system passing through the top sheet into the fluff pulpcomposition). In one embodiment the fluff pulp composition comprises aluciferin but not a luciferase. In one embodiment the fluff pulpcomposition comprises a luciferase but not a luciferin.

Fluff Pulp

The fluff pulp of the fluff pulp composition can be formed from anypulp. In one embodiment, the fluff pulp is derived from alignocellulosic fiber. In one embodiment, the fluff pulp is derived froma lignocellulosic fiber derived from wood. In one embodiment, the fluffpulp is derived from a lignocellulosic fiber derived from wood bychemical, mechanical, chemimechanical, or thermomechanical means. In oneembodiment, the fluff pulp is derived from a cellulosic fiber derivedfrom wood by chemical pulping. In one embodiment, the fluff pulp isderived from a cellulosic fiber derived from chemical pulping of woodeither by alcohol pulping, organo-solve pulping, acid sulfite pulping,alkaline sulfite pulping, neutral sulfite pulping, alkaline peroxidepulping, Kraft pulping, Kraft-AQ pulping, polysulfide pulping, orpolysulfide-AQ pulping.

In one embodiment, the fluff pulp is derived from a cellulosic fiberderived from chemical pulping of wood by further removing lignin fromthe said pulp either by alcohol pulping, organo-solve pulping, acidsulfite pulping, alkaline sulfite pulping, neutral sulfite pulping,alkaline peroxide pulping, Kraft pulping, Kraft-AQ pulping, polysulfidepulping, or polysulfide-AQ pulping for the preparation of absorbentarticles (fluff pulp). In one embodiment, the fluff pulp is derived froma cellulosic fluff pulp derived from Kraft pulping. In one embodiment,the fluff pulp is derived from a cellulosic bleached fluff pulp derivedfrom Kraft pulping. In one embodiment, the fluff pulp is derived from acellulosic bleached fluff pulp derived from Kraft pulping of softwoods.In one embodiment, the fluff pulp is derived from a cellulosic bleachedfluff pulp derived from Kraft pulping of Southern softwoods. In oneembodiment, the fluff pulp is derived from a cellulosic bleached fluffpulp derived from Kraft pulping of Southern pine. In one embodiment, thefluff pulp is derived from a Southern softwood. In one embodiment, thefluff pulp is derived from Southern pine.

The fluff pulp composition can be produced from pulp in any form, suchas a wet-laid sheet which is dried to achieve a moisture content rangingfrom about 6% to about 11% (e.g., 6% to 11%).

In another aspect, methods of preparing the fluff pulp composition areprovided. The fluff pulp composition is prepared by incorporating atleast one component of the chemiluminescent system into the fluff pulp.

This can be accomplished using various methods that allow the fluff pulpto be treated with one or more components of the chemiluminescentsystem. One challenge in the chemical treatment of fluff pulp is tomaintain the chemicals in a state in which the intended chemiluminescentreaction is not prematurely triggered, for example, before the treatedfluff pulp is incorporated into an absorbent article that is thensubjected to a liquid insult. For a wet end application, the chemicalstypically cannot be comingled with water and be applied together.Accordingly, in an illustrative example, either the luciferase orluciferin may be microencapsulated and introduced during the wet-layingprocess, with the non-encapsulated component applied to the sheet in anon-aqueous environment by standard methods such as coating, dipping,spraying, or printing (or combination thereof), prior to the air-laidoperation during absorbent article manufacture. In another illustrativeexample, a two sheet system, one containing luciferase and the othercontaining luciferin, may be made and processed further before theair-laid operation during absorbent article manufacture. In yet otherexamples, one of the chemicals maybe added during the wet-laying processand the other during the subsequent processing of the pulp; or the twocomponents may be added to the pulp during or prior to the air-laidprocess, such as by rinsing and/or spraying the pulp in fluffed formwith non-aqueous solutions of one or both the respective components.

Absorbent Articles

In one embodiment, the fluff pulp composition; the isolated compositionincluding the coelenterazine, or salt thereof; and/or thecoelenterazine, or salt thereof, can be incorporated into absorbentarticles. Representative absorbent articles include child diapers, adultdiapers, adult incontinence products, feminine hygiene products,absorbent underpads, and wound care dressing articles. For example, thefluff pulp composition and/or the coelenterazine, or salt thereof, canbe incorporated into one or more absorbent layers or portions of anabsorbent article.

In another aspect, an absorbent article is provided. In one embodiment,the absorbent article includes a top sheet that is liquid permeable, aback sheet that is liquid impermeable, fluff pulp disposed between thetop sheet and the back sheet and/or a fluffless or near flufflessnon-woven fabric matrix disposed between the top sheet and the backsheet, and a chemiluminescent system configured to produce light uponcontact with an aqueous system.

The chemiluminescent system (e.g., a luciferin such as coelenterazine,or a salt thereof of the present disclosure, and a luciferase) of theabsorbent article is as described herein. However, the chemiluminescentsystem need not be disposed, in whole or in part, within the fluff pulp.As discussed above, structural and fluid distribution functions may beprovided, in some configurations, by synthetic fibers, leading to thedevelopment of absorbent cores containing both fluff pulp fibers andsynthetic fibers, and even “fluff-less” absorbent cores containing nofluff pulp fibers. In some embodiments, the chemiluminescent system, orparts of the chemiluminescent system, can be independently integrated inthe liquid permeable top sheet, the liquid impermeable back sheet, theSAP, or another structure in the absorbent article.

In one embodiment, the chemiluminescent system comprises a luciferin anda luciferase. In one embodiment the luciferin and the luciferase areboth disposed within the fluff pulp. In another embodiment, one of theluciferin and the luciferase is disposed within the fluff pulp and theother is disposed in a different layer (e.g., top sheet or ADL) of theabsorbent product such that the two components are combined only when atleast one of the two components is carried by a liquid insult (e.g.,passing through the top sheet or ADL into the fluff pulp composition).In one embodiment the fluff pulp comprises a luciferin but not aluciferase. In one embodiment the fluff pulp comprises a luciferase butnot a luciferin.

In yet another embodiment, at least one component of thechemiluminescent system is disposed on (for example, printed onto) theinner surface of the backsheet.

In one embodiment, one of the luciferin and the luciferase is disposedwithin the fluff pulp and the other is associated with the top sheet oranother structure within an article, and configured to travel into thefluff pulp upon exposure to a liquid insult.

In one embodiment, the absorbent article further comprises a pH buffer,as disclosed herein. In one embodiment, the pH buffer is disposed withinthe fluff pulp. As discussed above, structural and fluid distributionfunctions may be provided, in some configurations, by synthetic fibers,leading to the development of absorbent cores containing both fluff pulpfibers and synthetic fibers, and even “fluff-less” absorbent corescontaining no fluff pulp fibers. In some embodiments, thechemiluminescent system, or parts of the chemiluminescent system, can beindependently integrated in the liquid permeable top sheet, the liquidimpermeable back sheet, the SAP, or another structure in the absorbentarticle.

In one embodiment, the absorbent article further comprises aphotoluminescent compound, as disclosed herein. In one embodiment, thephotoluminescent compound is disposed within the fluff pulp.

In one embodiment, the absorbent article further comprises aphotoluminescent compound and a pH buffer, as disclosed herein. In oneembodiment, the photoluminescent compound and the pH buffer are disposedwithin the fluff pulp.

In one embodiment, the pH buffer, the photoluminescent compound, theluciferin, and the luciferase are disposed within the fluff pulp.

In one embodiment, at least one of the pH buffer, the photoluminescentcompound, the luciferin, and the luciferase are not disposed within thefluff pulp. In some embodiments, at least one of the pH buffer, thephotoluminescent compound, the luciferin, and the luciferase can beindependently incorporated into synthetic fibers, “fluff-less” absorbentcores containing no fluff pulp fibers, in the liquid permeable topsheet, the liquid impermeable back sheet, and/or the SAP of theabsorbent article. In some embodiments, the pH buffer, thephotoluminescent compound, the luciferin, and/or the luciferase canmigrate to an absorbent core from a different structure of the article.In some embodiments, the pH buffer, the photoluminescent compound, theluciferin, and/or the luciferase can migrate from an absorbent core to adifferent structure of the article.

In one embodiment, the absorbent article further includes asuperabsorbent polymer (SAP), such as incorporated in the absorbentcore. In such an embodiment, at least one component of thechemiluminescent system may be disposed in the SAP, such that thechemiluminescence is generated upon the fluid from an insult travelingto the absorbent core.

In one embodiment the chemiluminescent system is contained entirelywithin an absorbent core of the absorbent article. As the absorbent coreis almost always a multi-component system there exist more than oneapproach to incorporate the chemiluminescent system into the absorbentcore. For instance the fluff pulp fibers could be the carrier of thechemiluminescent system. Alternatively, the chemiluminescent systemcould be contained within superabsorbent particles incorporated into theabsorbent article. In some embodiments, the absorbent core can includecellulose fibers, cellulose fiber derivatives (rayon, lyocell, etc.),nonwoven cellulose fibers and/or cellulose fiber derivatives,non-cellulose fibers, or any combination thereof.

Furthermore, if only a portion of the SAP particles or fibers containedthe chemiluminescent system chemistry, a desired pattern, such as anaesthetically pleasing pattern, can be achieved.

The chemiluminescent system can be added to the fluff pulp fibers at thetime of absorbent article manufacture or during an upstream processentirely separated from final product assembly. As noted above, forexample, the chemiluminescent system may be sprayed onto or otherwiseincorporated into a fluff pulp sheet prior to hammermilling.

In another aspect, methods of manufacturing absorbent articles are alsoprovided.

Absorbent articles are manufactured according to general techniquesknown to those of skill in the art that allow the incorporation of thechemiluminescent system to be incorporated into the absorbent article inthe manner disclosed herein.

The following examples are intended to be illustrative, not limiting.Example 1 describes the synthesis of coelenterazine on a 1 Kg scale.Example 2 describes the synthesis of coelenterazine on a 25 gram scale.Example 3 describes the synthesis of coelenterazine on a 100 gram scale.Example 4 describes the synthesis of coelenterazine on a multi-kilogramscale.

EXAMPLES Example 1. Synthesis of Coelenterazine (1 Kilogram) Synthesisof 3-benzylpyrazin-2-amine (25)

A 100 L round bottom flask was charged with 10 L of toluene followed by10 L tetramethylethylenediamine (TMEDA). The mixture was cooled at 0-5°C. with ice water. To this stirred mixture was added 20 L of n-butyllithium (2.5 M in n-hexane) solution at 0° C. to 8° C. drop wise over1-1½ hrs under nitrogen. After the complete addition, the reactionmixture was allowed to come to room temperature (22° C.). After 20minutes at room temperature, the mixture slowly heated up to 60° C.(During this time butane gas was released). The reaction mixture wasmaintained at 60° C.±1° C. for 30 minutes and then allowed to cool toroom temperature.

Meanwhile another 30 L flask was charged with tetrahydrofuran (THF) (15L) and pyrazin-2-amine (24) (also named 2-amino pyrazine (24)) (1 Kg).The mixture was stirred at 25-35° C. for 20 minutes. This solution wasadded over 1-1.5 hrs to the above benzyl lithium solution using additionflask. The mixture was stirred at room temperature for 1 hr. Water (16L) was added to the reaction mixture at 20° C. dropwise whilemaintaining the temperature between 20-25° C. The mixture was stirred atroom temperature for 30 min, and then the organic layer was separated,and distilled off excess solvents. The residue was treated with toluene(8 L) and water (3 L) and stirred for 10 minutes. The organic layer wasseparated, and solvent was distilled off at reduced pressure to yieldthe desired 3-benzylpyrazin-2-amine (25) in 60%-65% yield. Purity94%-95%.

Synthesis of 3-benzyl-5-bromopyrazin-2-amine (2)

A 20 L round bottom flask was charged with chloroform (6 L) and3-benzylpyrazin-2-amine (25) (also named 2-amino-3-benzyl pyrazine,(25)) (1 Kg), and stirred the mixture at room temperature (22° C.).N-bromosuccinimide (NBS) (800 grams) was added slowly over 1 to 1.5 hrs.After the complete addition, the mixture was stirred for 30 minutes.Water (2 L) was added and stirred for 10 minutes. The organic layer wasseparated and washed with water (2×1 L). The chloroform layer wasconcentrated under reduced pressure and the oily residue was dried undervacuum. Yield 77%-85%. Purity 93%-95%.

Synthesis of 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5)

A 50 L flask was charged with 1,4-dioxane (30 L) and3-benzyl-5-bromopyrazin-2-amine (2) (1 Kg) at room temperature.Potassium carbonate (1.6 Kg) was added followed by water (5 L). Thereaction mixture was stirred for 10 minutes. 4-methoxyphenyl boronicacid (4 L) (600 grams) was added followed by palladium catalyst (300grams). The reaction mixture was slowly heated up to 82° C. and stirredat 80-82° C. for 5 hrs. The mixture was cooled to room temperature andtransferred to 100 L round bottomed flask, and charged with ethylacetate (15 L) and water (15 L) at room temperature and stirred for 20minutes. The organic layer was separated and concentrated under reducedpressure to yield the desired product. Yield 85%-90%. Purity 92%-95%.

Synthesis of 4-(5-amino-6-benzyl-pyrazin-2-yl)phenol (7)

A 30 L round bottom flask was charged with3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (5) (1 Kg), and pyridinehydrochloride (6) (6.5 Kg). The reaction mixture was slowly heated to200° C. in an oil bath. The reaction temperature was maintained at200-210° C. for 30 min. The mixture was cooled to 40° C. and chargedwith water (13 L) and ethyl acetate (15 L) at 35-40° C. The reactionmixture was stirred for 20 minutes. The ethyl acetate layer wasseparated and the aqueous layer was re-extracted with ethyl acetate (3L×2). The combined ethyl acetate extracts were concentrated underreduced pressure. The residue was taken up with 2 L of ethyl acetate andrefluxed. The solution was slowly cooled to 10-15° C. and the solidformed was filtered to yield the desired product. Yield 90%-95%. Purity90%.

Alternatively, instead of using pyridinium chloride, a 30 L round bottomflask was charged with 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (1Kg), NaH (1 Kg), DMF (20 L) and ethanethiol (2 Kg). The reaction mixturewas slowly heated to 100° C. in an oil bath. The reaction temperaturewas maintained at 100-110° C. for 30 min. The mixture was cooled to 40°C. and charged with water (13 L) and ethyl acetate (15 L) at 35-40° C.The reaction mixture was stirred for 20 minutes. The ethyl acetate layerwas separated, and the aqueous layer was re-extracted with ethyl acetate(3 L×2). The combined ethyl acetate extracts were concentrated underreduced pressure. The residue was taken up with 2 L of ethyl acetate andrefluxed. The solution was slowly cooled to 10-15° C. and the solidformed was filtered to yield the desired product. Yield 90%-95%. Purity90%.

In both scenarios, the product can be optionally purified by charging 1Kg of the product and 5 L 1,4-dioxane at room temperature into a 10 Lround bottom flask, adding sodium hydroxide solution (250 g sodiumhydroxide in 1 L water) to the solution, charging the flask with 100 gof charcoal and 100 g silica gel at room temperature, stirring themixture at room temperature for 20 mins, and filtering and washing thesolids with 250 ml 1,4-dioxane. The filtrate was then transferred toanother 20.0 L round bottom flask and 200 ml HCl slowly added to thereaction mixture to adjust the pH to 7 to 7.5 at room temperature,during which a solid precipitate was observed. The reaction mixture wasthen stirred at room temperature for 30 mins, filtered, and washed with200 ml 1,4-dioxane. The isolated solid product was dried under vacuum,and in in the oven at 50-55° C. for 4 hrs. Yield 90%-95%. Purity 90%.

Synthesis of 4-(tert-butyldimethylsiloxy) benzyl alcohol (20b)

A 20 L round bottom flask was charged with dichloromethane (10 L),4-hydroxy benzaldehyde (8) (1 Kg). N,N′-dimethylaminopyridine (50 grams)and imidazole (1.33 Kg). The reaction mixture was cooled to 20° C. andstirred. To this stirred mixture was added portion wisetert-butyldimethylsilyl chloride (TBDMS-Cl, 500 grams×3). After 1 hour,the reaction mixture was filtered, and concentrated under reducedpressure to get an oily product (20a).

The above product (20a) was taken in a 10 L round bottom flask anddissolved in methanol (6 L). The reaction mixture was cooled to 10-15°C., and sodium borohydride (100 grams) was added with stirring. After 30minutes the reaction pH was adjusted to 7.0 with acetic acid. Afterstirring for 20 minutes, methanol was distilled off to yield the desired4-(tert-butyldimethylsiloxy) benzyl alcohol (20b). Yield 85%-90%, Purity80%-85%.

Synthesis of 4-(tert-butyldimethylsiloxy) benzyl chloride (21)

A 10 L round bottom flask was charged with4-(tert-butyldimethylsiloxy)benzyl alcohol (20b) (1 Kg) anddichloromethane (6 L) followed by triethylamine (1.4 L). The reactionmixture was stirred for 30 minutes at room temperature. Methanesulfonylchloride (600 mL) was added slowly at 30-35° C. in about 1-1.5 hours.After the completion of the reaction 30% aqueous sodium bicarbonatesolution (400 ml) was added and stirred for 20 minutes. Dichloromethanelayer was separated and washed with aqueous sodium chloride solution(2×500 ml). Dichloromethane was removed under reduced pressure. Theresidue was used for the next step without further purification.

Synthesis of3-(4-((tert-butyldimethylsilyfloxy)phenyl)-1,1-diethoxypropan-2-one (23)

A 50 L round bottom flask was charged with magnesium turnings (1 Kg) andanhydrous tetrahydrofuran (3 L) followed by iodine (10 grams) anddibromoethane (50 ml). A solution oftert-butyl(4-(chloromethyl)phenoxy)dimethylsilane (21) (1.6 Kg) inanhydrous tetrahydrofuran (12 L) was added drop wise at 40-45° C. over aperiod of 4 hours. The reaction mixture was cooled to 35° C. Another 50L round bottomed flask was charged with ethyl 2,2-diethoxyacetate (12)(2 Kg) and anhydrous tetrahydrofuran (10 L) and cooled to −35° C. Theabove prepared Grignard reaction mixture was added to this solution at−35° C. over a period of 1-1.5 hours. After the completion of thereaction, the reaction mixture was quenched with saturated aqueousammonium chloride solution (1.2 Kg in water 7 L). The organic layer wasseparated, washed with saturated sodium chloride solution and thesolvent was removed under reduced pressure. The oily residue waspurified by column chromatography over silica gel with an eluant thatincludes triethylamine to yield3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one (23)(0.48 Kg) Yield 20%-25%. Purity 90%.

Synthesis of coelenterazine8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one

A 30 L round bottom flask was charged with4-(5-amino-6-benzyl-pyrazin-2-yl)phenol (7) (0.9 Kg) and3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one (23)(1.4 Kg) followed by 1, 4-dioxane (14 L). The reaction mixture wasstirred at room temperature for 30 minutes. Concentrated hydrochloricacid (0.75 L) and water (0.75 L) was added and the reaction mixture washeated to 80-85° C. for 15 hours. The reaction mixture is cooled to 40°C. then activated carbon (100 g) and activated silica gel (100 g) areadded and filtered. The solvent was removed under reduced pressure andthe residue was precipitated by stirring with degassed ethyl acetate (2L). Yield 60%-65%. Purity 60%-63%.

Example 2. Synthesis of Coelenterazine (16)3-Benzyl-5-bromopyrazin-2-amine (2)

Zn dust and 30-mesh granular zinc (1:1, 16.05/16.05 g, 491.2 mmol, 3.5equiv) and 12 (6.23 g, 5% mol of Zn) were added to a dry 1 L 2-neckedround-bottom flask under an argon atmosphere. N,N-dimethylacetamide (125mL, freshly distilled over CaH₂) was added. The mixture was stirred atroom temperature until the brown color of the I₂ disappeared. Benzylbromide (61.04 g, 356.9 mmol, 2.5 equiv) was added dropwise, and themixture was stirred at 80° C. for 4 h. The mixture was cooled to roomtemperature and a suspension of 3,5-dibromo-2-aminopyrazine (1) (36.0 g,140.3 mmol, 1 equiv) and PdCl₂(PPh₃)₂ (5.04 g, 0.712 mmol, 5% ofpyrazine) in N, N-dimethylacetamide (150 mL) was added. The mixture wasstirred continuously under an argon atmosphere for 1 day. The thin layerchromatography (TLC) (30% ethyl acetate/hexane) indicated that thereaction was complete. The reaction mixture was filtered through a shortbed of celite. The filtrate was slowly poured into ice cold water (1 L),and extracted with EtOAc (3×200 mL). The combined organic layer waswashed with water (200 mL), brine (200 mL), and dried over anhydrousMgSO₄. The organic layer was filtered and concentrated on a rotaryevaporator under reduced pressure. The purification of the brown residueon a silica gel column chromatography eluting with hexane/EtOAc, 2:1gave 3-benzyl-5-bromopyrazin-2-amine (2) as brown viscous oil, 28.0 g74% yield. 1H NMR (CDCl₃): δ 8.05 (s, 1H), 7.22-7.35 (m, 5H), 4.41 (s,2H), 4.11 (s, 2H), 4.08 (s, 2H).

Palladium Coupling Reaction of p-methoxyphenyl Boronic Acid (4 L) with3-benzyl-5-bromopyrazin-2-amine (2)

1,4-bis(diphenylphosphino)butane (2.71 g, 6.34 mmol) was added to asuspension of bis(benzonitrile)dichloropalladium (II) (2.03 g, 5.29mmol) in toluene (210 mL) at room temperature under an argon atmosphereand stirred for 30 minutes. A solution of3-benzyl-5-bromopyrazin-2-amine (2) (28.0 g, 106.0 mmol) in toluene (180mL) was added to this mixture followed by 4-methoxyphenylboronic acid(20.94 g, 137.8 mmol), ethanol (42 mL), and 1.0 M aq. Na₂CO₃ (108 mL)were added sequentially at room temperature with stirring. The resultingmixture was heated to reflux for 4 h and then allowed to cool to roomtemperature. The mixture was diluted with 20% aq. NaCl solution (400 mL)and extracted with ethyl acetate (EtOAc, 3×300 mL). The combined organiclayer was washed with water (200 mL), brine (200 mL), and dried overanhydrous MgSO₄. The organic layer was filtered and the ethyl acetatesolution was treated with 2 N HCl (200 mL) to precipitate the product asits hydrochloride salt. A yellow precipitate formed immediately. Theprecipitated solid was isolated by filtration and dried under vacuum.The dried solid was washed with ethyl acetate (2×100 mL) to remove nonpolar impurities and subsequently dried under vacuum to yield3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine hydrochloric acid salt (5),24 g, 78% yield. ¹H NMR (CDCl₃): δ 8.05 (s, 1H), 7.22-7.35 (m, 5H), 4.41(s, 2H), 4.11 (s, 2H), 4.08 (s, 2H). The LCMS analysis indicated thatthe product was around 99% pure.

De-Methoxylation of Methyl Ether Hydrochloride Salt to Phenol UsingPyridinium Hydrochloride

A mixture of 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine hydrochloricacid salt (5) (20.0 g, 61.0 mmol) and pyridinium hydrochloride (6) (70.5g, 0.61 mol) was heated at 200° C. for 2 h under an argon atmosphere.The dark brown colored mixture was cooled to room temperature, and asaturated sodium bicarbonate solution (750 ml) was added to the solidslowly followed by ethyl acetate (750 ml)). The organic layer wasseparated, and the aqueous layer was and extracted with EtOAc (3×200mL). The combined ethyl acetate extracts were washed with water (2×300mL), dried over anhydrous MgSO₄. The solution was filtered andconcentrated on a rotary evaporator under reduced pressure. Purificationof the brown reside on a silica gel column chromatography eluting withhexane-EtOAc, (3:7) gave 4-(5-amino-6-benzyl-pyrazin-2-yl)phenol (7), 14g, 82% yield as light yellow solid., The solid was recrystallized fromethyl acetate/1% methanol to give a light brown powder. ¹H NMR (DMSO-D6)δ 9.36 (s, 1H), 8.17 (s, 1H), 7.60 (d, J=8.4 Hz, 2H), 7.13-7.22 (m, 5H),6.68 (d, J=8.6 Hz, 2H), 6.09 (s, 2H), 3.94 (s, 2H). The LCMS analysis ofthe product indicated that it was around 99% pure.

4-Benzyloxybenzaldehyde (9)

A mixture of 4-hydroxybenzaldehyde (8) (122.1 g, 1.0 mol), benzylchloride (132.9 g, 1.05 mol), and anhydrous potassium carbonate (165.6g, 1.2 mol) in N,N-dimethyl formamide (DMF, 1 L) was heated at 70-80° C.for 3 days with vigorous stirring. The progress of the reaction wasmonitored by TLC (20% ethyl acetate/hexane). The mixture was poured intoice cold water (4 L). The resulting solid was collected by filtration,washed with water (2×500 ml) and dried to yield the desired product as awhite solid. Wt=210 g, and yield 96.3%.

4-Benzyloxybenzyl Alcohol (10)

Sodium borohydride (40.0 g, 1.08 mol) was slowly added portion wise to asolution of 4-(benzyloxy)benzaldehyde (9) (218.0 g, 1.02 mmol) in amixture of ethyl acetate/methanol (1:1, 1 L) at 0-5° C. over a period of1 h. After the complete addition of sodium borohydride, the reactionmixture was slowly warmed to room temperature and stirred for 2 h. Theprogress of the reaction was monitored by TLC (20% ethylacetate/hexane). The mixture was concentrated under reduced pressure,and the residue was partitioned between ethyl acetate (750 ml) and water(500 ml). The organic layer was separated. The aqueous layer wasextracted with ethyl acetate (2×200 ml). The combined ethyl extractswere washed with water (500 ml) and dried over anhydrous MgSO₄,filtered, and concentrated under reduced pressure. The residue wasrecrystallized with 20% ethyl acetate/hexane to yield desired product.Wt=180.5 g, and yield 82.7%.

4-Benzyloxybenzyl chloride (11)

a) Using Thionyl Chloride.

Thionyl chloride (101.2 g, 0.86 mol) was added drop wise to a cooledsolution of (4-(benzyloxy)phenyl)methanol (10) (167.0 g, 0.78 mol) inethyl acetate (600 ml) at 0° C. over 1 h. After the addition thereaction mixture was warmed to room temperature and stirred. Theprogress of the reaction was monitored by TLC, after 4 h reaction wascomplete. The reaction mixture was concentrated under reduced pressure(<50° C.). The resulting residue was recrystallized twice with hexane toyield the desire product as white solid. Wt=180 g, yield 77%. ¹H NMR(400 MHz, CDCl3): δ=4.57 (s, 2H), 5.08 (s, 2H), 6.96 (d, J=8.7 Hz, 2H),7.32 (d, J=8.7 Hz, 2H), 7.43-7.37 (m, 5H).

b) Using Cyanuric Chloride:

Cyanuric chloride (1.0 g) was added to anhydrous N,N′-dimethylformamide(5 ml) at room temperature under an atmosphere of argon and stirred for30 minutes. A white suspension was formed. A solution of(4-(benzyloxy)phenyl)methanol (1.0 g) in dichloromethane (30 ml) wasadded and stirred overnight. The precipitated solids were removed byfiltration. The filtrate was diluted with hexane (50 ml) and washed withwater, brine solution (20 ml each), dried over anhydrous magnesiumsulfate. The solvent was removed under reduced pressure. The product wasfurther purified by column chromatography over silica gel eluting with5% ethyl acetate/hexane to give the product, Wt 650 mg Yield=59.5%

c) Using Methanesulfonyl Chloride:

Methanesulfonyl chloride (13.74 g; 0.12 mol) was added dropwise to asolution of (4-(benzyloxy)phenyl)methanol (21.8 g; 0.10 mol) andtriethylamine (15.15 g; 0.15 mol) in dichloromethane (250 ml) at 0° C.After the addition the reaction mixture was warmed to room temperatureand stirred 12 h. The solvent was removed under reduced pressure. Theresidue was partitioned between ethyl acetate (200 ml) and water (100ml). The ethyl acetate layer was separated, washed with water, brinesolution (75 ml each), dried over anhydrous magnesium sulfate. The crudeproduct obtained after the removal of the solvent was recrystallizedfrom hexane to obtain 18 gram of the product. Wt=18 grams (Yield 70%).

3-[4-(Benzyloxy)phenyl]-1,1-diethoxypropan-2-one (13)

Mg turnings (10.57 g, 0.435 mole, 1.5 equiv) were suspended in drydistilled THF (100 mL) in an argon-flushed, 500 mL two neckedround-bottom flask. A solution of (11) (68.14 g, 0.29 mole) in THF (600ml) was prepared and 25 ml of the solution was added at once and theflask was warmed to 40-50° C. with constant stirring until the Grignardreaction was initiated. After the initiation the remaining solution wasadded slowly at a rate so that the reaction mixture was warm to touchdue to the heat of the reaction. After the addition mixture was stirredat room temperature for 30 min and then refluxed for 1 hour to completethe reaction. The pale yellow Grignard reagent was allowed to cool toroom temperature and was then kept in an ice bath. A solution of ethyldiethoxy acetate (12) (51 g 0.29 mole,) in THF (200 mL) in a separate 1liter round-bottom flask under an argon atmosphere and cooled to −78° C.The Grignard reagent was transferred to dropping funnel and was addeddrop wise into the cooled flask over 45 min. The mixture was thenstirred for 6 h at −78° C. and warmed to −20° C. and stirred for 2 h.The reaction was quenched with saturated ammonium chloride solution (200mL). The reaction mixture was extracted with ethyl acetate (500 mL). Theethyl acetate layer was washed with H₂O (3×200 mL), followed bysaturated brine solution (2×200 mL). The organic layer was dried(anhydrous magnesium sulfate) and the solvent was removed by rotaryevaporation under reduced pressure. The product was heated to 150° C.under reduced pressure to remove the impurities and unreacted startingmaterials. NMR of the product showed it to be sufficiently pure to carryout the next reaction. Wt=66.58 g Yield (70%)

¹H NMR (400 MHz, CDCl₃): δ=1.25 (t, J=7.3 Hz, 6H), 3.57 (m, 2H), 3.69(m, 2H), 3.84 (s, 2H), 4.64 (s, 1H), 5.05 (s, 2H), 6.94 (d, J=8.4 Hz,2H), 7.14 (d, J=8.4 Hz, 2H), 7.43-7.35 (m, 5H).

1,1-Diethoxy-3-(4-hydroxyphenyl)-2-propan-2-one) (14)

Initial attempts to debenzylate under an atmosphere of hydrogen usingpalladium 10% on charcoal without the pressure reactor were notsuccessful.

A solution of the compound (13) (66 g, 20.4 mole) in ethanol (400 mL)was placed in a Parr hydrogenation flask and 10% Pd/C (7 g) was added.The mixture was hydrogenated for 24 hours under hydrogen atmosphere at60 Psi. The black suspension was filtered and the solvent was removedusing a rotary evaporator. The product was purified by passing through asmall pad of silica gel to remove any suspended carbon particles elutingwith 50% ethyl acetate/hexane. The desired product was isolated over asilica gel column eluting with 30% ethyl acetate/hexane to a colorlessoil, 21.7 g.

¹H NMR (400 MHz, CDCl₃): δ=1.25 (t, J=7.0 Hz, 6H), 3.55 (m, 2H), 3.71(m, 2H), 3.82 (s, 2H), 4.64 (s, 1H), 5.11 (br s, 1H), 6.77 (d, J=8.6 Hz,2H), 7.07 (d, J=8.6 Hz, 2H).

Coelenterazine[8-Benzyl-6-(4-hydroxyphenyl)-2-[(4-hydroxyphenyl)methyl]imidazo[1,2-α]pyrazin-3(7H)-one (16)

The effect of dry hydrochloric acid, organic acids, vs. aqueoushydrochloric acid on the final condensation, rearrangement, andcyclization reaction of coelenterazine were evaluated. The followingexperiments were conducted either in ethanol or 1,4-dioxane. The resultsare summarized in the following Table 1.

TABLE 1 Coupling reaction conditions.

Reaction conditions Result* 1 equivalent (eq) 1 eq Ethanol (EtOH)/dry —HCl, 80° C., 8 h 1 eq 1 eq EtOH/p- — Toluenesulfonic acid (PTSA), 80°C., 8 h 1 eq 1 eq EtOH/Trifluoroacetic — acid (TFA), 80° C., 8 h 1 eq 1eq EtOH/Conc. HCl, ~50% 80° C., 8 h conversion 1 eq 1 eq Dioxane/Conc.HCl, Reaction is 80° C., 8 h better than ethanol *The reaction mixturewas analyzed by reverse phase HPLC. An authentic coelenterazine samplewas used to identify the desired product.

The results from the above experiments (Table 1) indicated that thefinal condensation, rearrangement, and cyclization reaction ofcoelenterazine requires aqueous hydrochloric acid and the desiredsolvent is 1,4-dioxane, which proved to be better than ethanol.

After finding right solvent and acid, experiments were conducted withvarying amounts of starting material (14). Starting material (15) waskept constant at 1 equivalent and the amount of starting material (14)was increased. Unreacted excess of the reagent (starting material 14)could be removed by washing with organic solvent. Furthermore, underacidic reaction conditions only pyrazinamine starting material (15)derived product will form salt but not the acetal (14). To obtaininsight into the correct amount of reagents required for optimalreaction conditions, the following experiments were conducted. Theresults are summarized in the following Table 2.

TABLE 2 Coupling reaction starting material ratios.

Reaction conditions Result* 1 eq   1 eq Dioxane (0.6 Good ml)/6N HClreaction, (140 mg), some side water (0.1 product ml), 80° C., overnight1 eq 1.3 eq Dioxane (0.6 Better ml)/6N HCl reaction, (140 mg), minimumwater (0.1 side ml), 80° C., products overnight 1 eq 1.7 eq Dioxane (0.6Best ml)/6N HCl reaction, (140 mg), minimum water (0.1 side ml), 80° C.,products overnight *The reaction mixture was analyzed by reverse phaseHPLC. An authentic Coelenterazine sample was used to identify thedesired product.

Coelenterazine, [8-benzyl-6-(4-hydroxyphenyl)-2-[(4-hydroxyphenyl)methyl]imidazo[1,2-α]pyrazin-3(7H)-one (16)

1,4-dioxane (2.3 mL), water (225 μL), and conc. HCl (225 μL) taken in a25 ml round bottomed flask was degassed and filled with argon.4-(5-amino-6-benzyl-pyrazin-2-yl)phenol ((7), 441 mg, 1.59 mmol) wasadded to this mixture. A solution of1,1-diethoxy-3-(4-hydroxyphenyl)-2-propan-2-one) ((14), 493 mg, 2.06mmol, 1.3 eq) in 1,4-dioxane (2 mL) was added to this mixture. Theresulting mixture was degassed and stirred under argon atmosphere at78-82° C. for 14 hr. The dark brown solution was cooled to roomtemperature, and an aliquot was analyzed by reverse phase HPLC. The HPLCanalysis indicated that the reaction mixture had little startingmaterial. The reaction was degassed, and heating was continued forfurther 6 h (total 20 h). The reaction mixture was cooled to roomtemperature, and concentrated under reduced pressure. The dark brownresidue was dried under high vacuum overnight. Ethyl acetate (40 mL) wasadded to the residue and triturated. The solid was allowed to settledecanted, and dried under vacuum to yield brown dry powder. Degassedethyl acetate (40 mL) containing 1% methanol was added to the brownsolid and triturated at 65° C. The solid was allowed to settle, anddecanted, and dried at pump to yield brown dry powder, 720 mg,quantitative yield. The proton NMR data is identical with that ofreported values. On reverse phase HPLC it co-eluted with an authenticsample. The solid was again suspended in 40 ml of degassed ethyl acetatecontaining 1% methanol and stirred for 15 min., and decanted.

The sample was dried at pump to yield brown solid.

TABLE 3 Mass spectra and LCMS analysis of 3 different samples. SampleCoelenterazine Coelenterazine amine Unknown 1 62% 11% 5.4% 2 65% 11%6.2% 3 66% 10% 6.3%

Coelenterazine, [8-Benzyl-6-(4-hydroxyphenyl)-2-[(4-hydroxyphenyl)methyl]imidazo[1,2-α]pyrazin-3(7H)-one (16): 25 g scale synthesis

1,4-dioxane (80.0 mL), water (9.1 mL), and conc. HCl (9.1 mL) taken in300 ml round bottomed flask was degassed and filled with argon.4-(5-amino-6-benzyl-pyrazin-2-yl)phenol (7, 15.94 g, 59.93 mmol) wasadded to this mixture and stirred . a degassed 1,4-dioxane (71 mL)solution of 1,1-diethoxy-3-(4-hydroxyphenyl)-2-propan-2-one) (14, 16.4g, 68.82 mmol, 1.2 eq) was added to this suspension. The resultingmixture was degassed and stirred under argon atmosphere at 78-84° C. for34 h (the reaction progress was monitored by reverse phase HPLC). Thereaction was cooled to room temperature, and concentrated under reducedpressure. The dark brown residue was dried under high vacuum overnight.Degassed ethyl acetate (250 mL) was added to this brown residue andtriturated. The solid was allowed to settle, and decanted. This processwas repeated second time, and the solid was dried at pump for 24 h toyield a brown dry powder, 25.8 g, in quantitative yield.

Example 3. Synthesis of Coelenterazine HCl Salt, 100 g Scale StartingMaterial Synthesis:

The starting material 4-(5-amino-6-benzyl-pyrazin-2-yl)phenol (7) (53.0g), and 1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one (14) (57.0 g) weresynthesized by following the procedure described previously in Example2.

8-Benzyl-6-(4-hydroxyphenyl)-2-[(4-hydroxyphenyl)methyl]-7H-imidazo[1,2-α]pyrazin-3-one(3)

A 1 L round bottom flask equipped with a stirrer bar was charged with4-(5-amino-6-benzyl-pyrazin-2-yl)phenol (7) (53.0 g, 191.25 mmol),followed by 1,4-dioxane (275 mL). The resulting mixture was degassed andfilled with argon. To this stirred mixture was added degassed 1:1water/conc. HCl (62.0 mL). The resulting mixture was again degassed andfilled with argon, and stirred at room temperature for 15 min. To thisstirred suspension was added a degassed 1,4-dioxane (244 mL) solution of1,1-diethoxy-3-(4-hydroxyphenyl)-2-propan-2-one (14) (57.0 g, 239.21mmol, 1.25 eq). The resulting mixture was degassed and stirred underargon atmosphere at 80-85° C. for 38 h (the reaction progress wasmonitored by reverse phase HPLC).

The reaction was cooled to room temperature, and concentrated underreduced pressure. The resulting dark brown residue was dried under highvacuum overnight. To this was added degassed ethyl acetate (250 mL) andtriturated. The solid was allowed to settle, and decanted. This processwas repeated one more time. Then added a degassed ethyl acetate (200 mL)containing 1% methanol to the brown solid and triturated. The solid wasallowed to settle, and decanted. The residual dark brown solid was driedat high vacuum at 40° C. for 36 h, and stored under argon atmosphere,yield 101.8 g. On reverse phase HPLC it co-eluted with an authenticsample. The proton NMR spectral data were consistent with the reportedvalues.

Example 4. Synthesis of Coelenterazine (Multiple Kilograms) Synthesis of3-benzylpyrazin-2-amine (25)

A 100 L round bottom flask was charged with 7.5 L of THF, 2.5 kg ofmagnesium metal, 10 g of iodine, and 200 ml ethyl bromide. The reactionmass was initiated. After initiation, benzyl chloride in THF solution(10 L benzyl chloride dissolved in 45 L of THF) was slowly added at20-25° C. over a period of 4 to 4.5 hours and thereafter maintained forone hour at 30-35° C. Then, 2-amino pyrazine solution (2.5 kg 2-aminopyrazine dissolved in 25 L THF at 30-35° C. in 1 to 1.5 hours) wasslowly added and the reaction mass was maintained for 5-6 hours at30-35° C. A TLC check was done, after compliance 10 L water was slowlyadded and the reaction mass was stirred for 20 minutes. The reactionmass was then allowed to settle for 30 minutes. The THF layer wasseparated and distilled out completely under vacuum below 70° C. Aftercompleting the distillation, the reaction mass was then cooled to roomtemperature and charged with 20 L toluene and 5 L water and stirred for10 minutes. The reaction mass was then allowed to settle for 20 minutesand the toluene layer was separated. The toluene layer was then chargedwith 3 L HCl and allowed to settle for 10 minutes. Then the toluenelayer was separated and kept aside. The acidic HCl layer was thenadjusted to a pH of about 8-9 with 3 kg soda ash and maintained for 30minutes. The organic layer was then separated to yield the desired monoalkylated product in 40%-45% yield, purity 94%-95%.

The present modification reduces or eliminates the use of: n-butyllithium-in the first step of the synthesis of Example 1, where n-butyllithium reaction in toluene has been replaced by reaction with benzylchloride and THF (tetrahydrofuran). Thus, the present Example improvesthe ability to scale up the reaction chemistry and reduces the cost ofthe synthesis. In addition, the changes improve the overall safety ofthe chemistry by replacing highly reactive materials with more stablematerials.

Synthesis of 3-benzyl-5-bromopyrazin-2-amine (2)

The synthesis of 3-benzyl-5-bromopyrazin-2-amine is as described above,in Example 1. Specifically, a 20 L round bottom flask was charged withchloroform (6 L) and 3-benzylpyrazin-2-amine (25) (also named2-amino-3-benzyl pyrazine, (25)) (1 Kg), and the mixture was stirred atroom temperature (22° C.). N-bromosuccinimide (NBS) (800 grams) wasadded slowly over 1 to 1.5 hrs. After the complete addition, the mixturewas stirred for 30 minutes. Water (2 L) was added and stirred for 10minutes. The organic layer was separated and washed with water (2×1 L).The chloroform layer was concentrated under reduced pressure and theoily residue was dried under vacuum. Yield 77%-85%. Purity 93%-95%.

Synthesis of 4-(5-amino-6-benzylpyrazin-2-yl)phenol (7)

2 kg of 3-benzyl-5-bromopyrazin-2-amine (2) was dissolved in 10 L THFand 20 g of palladium catalyst (significantly reduced from Example 1,1/15 of the 300 g of palladium catalyst in Example 1) and stirred atroom temperature for 30 minutes. This solution was added to the Grignardreagent prepared using para bromo-phenol 3 kg, 4 kg of TBDMS chloride(t-butyldimethylsilyl chloride), and 2.5 kg of Mg metal. The reactionmixture was heated to 50° C. for 24 hours. After the completion of thereaction providing (26), Mg metal was filtered and diluted HCl 3 L wasadded to reaction mixture and heated to 70° C. for 8 hours. To thisreaction mixture containing (26) was added 2 L of water and the productwas extracted with ethyl acetate 2 L, this was repeated three times. Thecombined ethyl acetate layers were washed with water 1.5 L, twice. Theethyl acetate is distilled to get the product (7) as a thick liquid witha weight of about 2.5 kg. Yield 70%-75% Purity 84%-88%.

Thus, the present example omits the use of boronic acid compounds in thesynthesis, and greatly reduces the amount of expensive palladiumcatalyst in the reaction.

Synthesis of 4-((tert-butyl(4-(chloromethyl)phenoxy)dimethylsilane

A 20 L round bottom flask was charged with dichloromethane (10 L),4-hydroxy benzaldehyde (8) (1 Kg). N,N′-dimethylaminopyridine (50 grams)and imidazole (1.33 Kg). The reaction mixture was cooled to 20° C. andstirred. To this stirred mixture was added portion-wisetert-butyldimethylsilyl chloride (TBDMS-Cl, 500 grams×3). After 1 hour,the reaction mixture was filtered, and concentrated under reducedpressure to get an oily product(4-((tert-butyldimethylsilyl)oxy)benzaldehyde, (20a)).

The above product (20a) was taken in a 10 L round bottom flask anddissolved in methanol (6 L). The reaction mixture was cooled to 10-15°C., and sodium borohydride (100 grams) was added with stirring. After 30minutes the reaction pH was adjusted to 7.0 with acetic acid. Afterstirring for 20 minutes, methanol was distilled off to yield the desiredproduct (20b). Yield 85%-90%, Purity 80%-85%.

A 10 L round bottom flask was charged with4-(tert-butyldimethylsiloxy)benzyl alcohol (20b) (1 Kg) anddichloromethane (6 L) followed by triethylamine (1.4 L). The reactionmixture was stirred for 30 minutes at room temperature. Methanesulfonylchloride (600 mL) was added slowly at 30-35° C. in about 1-1.5 hours.After the completion of the reaction 30% aqueous sodium bicarbonatesolution (400 ml) was added and stirred for 20 minutes. Dichloromethanelayer was separated and washed with aqueous sodium chloride solution(2×500 ml). Dichloromethane was removed under reduced pressure. Theresidue containing tert-butyl(4-(chloromethyl)phenoxy)dimethylsilane(21) was used for the next step without further purification.

Synthesis of3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-dimethoxypropan-2-one)(27)

A 50 L round bottom flask was charged with magnesium turnings (1 Kg) andanhydrous tetrahydrofuran (3 L) followed by iodine (10 g) anddibromoethane (50 mL). A solution oftert-butyl(4-(chloromethyl)phenoxy)dimethylsilane (21) (1.6 kg) inanhydrous tetrahydrofuran (12 L) was added drop wise at 40-45° C. over aperiod of 4 hours. The reaction mixture was cooled to 35° C. Another 50L round bottomed flask was charged with methyl 2,2-dimethoxyacetate (1.2kg) and anhydrous tetrahydrofuran (10 L) and cooled to 30-35° C. Theabove-prepared Grignard reaction mixture was added to this solution at−10° C. over a period of 1-1.5 hours. After the completion of thereaction, the reaction mixture was quenched with saturated aqueousammonium chloride solution (1.2 Kg in water 7 L). The organic layer wasseparated, washed with saturated sodium chloride solution and thesolvent was removed under reduced pressure. The oily residue waspurified by column chromatography over silica gel to yield3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-dimethoxypropan-2-one(27) (0.48 Kg) Yield 40%-45%. Purity 90%.

Synthesis of coelenterazine,8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one (16)

A 30 L round bottom flask was charged with4-(5-amino-6-benzyl-pyrazin-2-yl)phenol (7) (0.9 Kg) and3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-dimethoxypropan-2-one(27) (1.4 Kg) followed by 1, 4-dioxane (14 L). The reaction mixture wasstirred at room temperature for 30 minutes. Concentrated hydrochloricacid (0.75 L) and water (0.75 L) was added and the reaction mixture washeated to 80-85° C. for 15 hours. The reaction mixture was cooled to 40°C. then activated carbon (100 g) and activated silica gel (100 g) wereadded and filtered. The solvent was removed under reduced pressure andthe residue (16) was precipitated by stirring with degassed ethylacetate (2 L). Yield 60%-65%. Purity 60%-63%.

Example 5. LC-MS Characterization of Isolated CoelenterazineCompositions

The relative amount of coelenterazine to4-(5-amino-6-benzylpyrazin-2-yl)phenol (7) (coelenteramine) in the finalisolated composition from the final coupling reaction to formcoelenterazine in Examples 1-3 can be assessed by liquidchromatography-mass spectrometry (LC-MS).

A 1 mg/ml methanolic solution of isolated coelenterazine compositionsresulting from coupling reactions of4-(5-amino-6-benzyl-pyrazin-2-yl)phenol (7) and3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one(23), as described, for example in Example 1 above, was diluted tentimes into an injection solvent consisting of 70:30 reagentwater:acetonitrile (v/v), each supplemented with 0.05% formic acid. Thediluted solution including the isolated coelenterazine composition wasseparated by LC on a C-18 reverse phase column using a gradient elution.The separation provided a response for coelenterazine at about 1.7 minand a response for coelenteramine (7) at about 2.5 min. Tandem MS wasconfigured to monitor the (M+H)+ parent ion of each compound which wassubsequently fragmented into its characteristic daughter ion. Thedaughter ion intensity created the chromatographic signal for eachcompound which was then integrated to produce an area for the signal.The parent ion for coelenterazine is 424.1 Da with a daughter ion at302.2 Da. The parent ion for coelenteramine is 278.1 Da and its daughterwas 132.0 Da. In Table 4 below, the ratio of the coelenterazine tocoelenteramine in the isolated composition was between about 24:1 and80:1.

TABLE 4 Integrated peak ratios of coelenterazine to coelenterazine asassessed by LC-MS. Coelenter- Coelenterazine/ Coelenterazine aminecoelentera- integrated integrated amine Composition peak area peak areapeak ratio Isolated composition 1 3.63E+07 1.52E+06 24 Isolatedcomposition 2 3.15E+07 3.93E+05 80 Isolated composition 3 2.72E+075.46E+05 50 Isolated composition 4 2.87E+07 5.07E+05 53 Isolatedcomposition 5 2.91E+07 4.45E+05 65

Example 6. Synthesis of Coelenterazine using4-(5-amino-6-benzylpyrazin-2-yl)phenol and3-(4-(benzyloxy)phenyl)-2-oxopropanal 4-(5-Amino-6-benzyl-pyrazin-2-yl)phenol (coelenteramine) 3-Benzylpyrazin-2-amine

A 100 L round bottom flask was charged with 7.5 L of tetrahydrofuran(THF), 2.5 kg magnesium metal, 10 g of iodine, and 200 ml ethyl bromide.The reaction mass was initiated, benzyl chloride and THF solution (10 Lbenzyl chloride dissolved in 45 L of THF) was slowly added at 10-20° C.over a period of 4 to 4.5 hours and thereafter maintained for 1 hour at30-35° C. Then 2-amino pyrazine solution (2.5 kg 2-amino pyrazinedissolved in 25 L THF at 30-35° C. in 1 to 1.5 hrs) was slowly added andthe reaction mass was maintained for 5-6 hours at 30-35° C. A thin layerchromatography (TLC) check was done, after compliance 10 L water wasslowly added and the reaction mass was stirred for 20 minutes.

The reaction mass was then allowed to settle for 30 mins. The THF layerwas separated and distilled out completely under vacuum below 70° C.After completing the distillation, the reaction mass was then cooled toroom temperature and charged with 20 L toluene and 5 L water and stirredfor 10 minutes. The reaction mass was then allowed to settle for 20minutes and the toluene layer was separated. The toluene layer was thencharged with 3 L HCl and allowed to settle for 10 minutes. Then thetoluene layer was separated and kept aside. The acidic HCl layer wasthen adjusted to a pH of about 8-9 with 3 kg soda ash and maintained for30 minutes. The organic layer was then separated to yield the desiredmono alkylated product in 45-50% yield, at a purity 90-95%.

3-Benzyl-5-bromopyrazin-2-amine

A 20 L round bottom flask was charged with chloroform (6 L) and2-amino-3-benzyl pyrazine (1 Kg), and stirred the mixture at roomtemperature (22° C.). N-bromosuccinimide (800 grams) was added slowlyover 1-1.5 h. After the complete addition, the mixture was stirred for30 minutes. Water (2 L) was added and stirred for 10 minutes followed bythe separation of the chloroform layer. HCl (500 ml) was added to thechloroform layer and the mixture was stirred for about 20 minutes. Theproduct was filtered through Nutsche filter and was given a wash withchloroform (1 L). The product was dried at 35-40° C. for 6-7 hrs. Yield70-75%. Purity 90-95%.

3-Benzyl-5-(4-methoxyphenyl)pyrazin-2-amine

A 50 L flask was charged with 1, 4-dioxane (30 L) and3-benzyl-5-bromopyrazin-2-amine (1 kg) at room temperature. Potassiumcarbonate (1.6 kg) was added followed by water (5 L). The reactionmixture was stirred for 10 minutes. 4-Methoxy phenyl boronic acid (600grams) was added followed by palladium catalyst (40 grams). The reactionmixture was slowly heated up to 82° C. and stirred at 80-82° C. for20-24 h under nitrogen. The mixture was cooled to 40° C. and transferredto 100 L round bottomed flask, and charged with ethyl acetate (15 L) andwater (15 L) at room temperature and stirred for 20 minutes. The organiclayer was separated and concentrated under reduced pressure to yield thedesired product. Yield 85-90%. Purity 92-95%.

4-(5-Amino-6-benzyl-pyrazin-2-yl) phenol

A 30 L round bottom flask was charged with 3-benzyl-5-(4-methoxyphenyl)pyrazin-2-amine (1 Kg), 4 L of 48% HBr and 7 L acetic acid. The reactionmixture was slowly heated to 110° C. in an oil bath. The reactiontemperature was maintained at 108-110° C. for 8-10 hours. The mixturewas cooled to 40° C. and charged with water (15 L) and ethyl acetate (15L) at 35-40° C. The reaction mixture was stirred for 20 minutes. The pHof the mixture was adjusted to 4-4.5 using soda-ash and the mixture wasstirred for 10 minutes.

The ethyl acetate layer was separated, and the aqueous layer wasre-extracted with ethyl acetate (3 L×2). The combined ethyl acetateextracts were adjusted to pH 7-7.5 with soda-ash and was concentratedunder reduced pressure. The residue was first cooled to 40° C. and wasthen taken up with 4 L of cyclohexane and refluxed. The compound wasfiltered through Nutsche filter and dried at 40-45° C. for 5-6 hrs.Yield: 75-80% Purity: 90%.

3-(4-(benzyloxy)phenyl)-2-oxopropanal 4-(benzyloxy)benzaldehyde

A 50 L round bottom flask was charged with dimethylformamide (15 L),4-hydroxybenzaldehyde (2.5 Kg) and the mixture was stirred for 20minutes. To this mixture, potassium carbonate (4 Kg) was added and thereaction mass was stirred for 10 minutes after which benzyl chloride(2.5 L) was added slowly for approximately 15 minutes. The mixture wasstirred for 20 minutes and was slowly heated to 40° C. followed bystirring at 45-45° C. for 6-7 hrs. The mixture was cooled to 30° C. andcharged with water (25 L). The mixture was cooled further to 20° C. andstirred for 30 minutes. The product obtained was centrifuged and washedwith water until the product became pH neutral. The product was thendried at 30-35° C. for 5-6 hrs. Yield: 70-75% Purity: 85-90%.

(4-(benzyloxy)phenyl)methanol

A 50 L round bottom flask was charged with 4-(benzyloxy)benzaldehyde (4Kg) and methanol (15 L). To this mixture, sodium borohydride (0.5 Kg)was added drop-wise at 45-50° C. over 1-1½ hrs. After the addition wascompleted, the mixture was stirred for 30 minutes. The reaction mixturewas cooled to 15° C. and was charged slowly with aqueous acetic acid(500 mL acetic acid in 500 mL water) followed by slow addition of water(25 L). This mixture was then stirred for 30 minutes at 15-20° C. Theproduct was centrifuged and dried at 40-45° C. over 12-14 hrs. Theresultant product was charged with n-hexane (10 L) and the reactionmixture was heated slowly to 50° C. The mixture was stirred at 50° C.for 1 hr after which it was cooled to 40° C. The product,(4-benzyloxy)phenyl)methanol was filtered through a

Nutsche filter and it was then dried at 40-45° C. over 12 hrs. Yield:85-90% Purity: 90-95%.

1-(benzyloxy)-4-(chloromethyl)benzene

A 50 L round bottom flask was charged with dichloromethane (20 L) and(4-(benzyloxy)phenyl)methanol (3.8 Kg) at room temperature. The mixturewas stirred for 20 minutes and was charged with dimethylformamide (500mL) followed by stirring for another 10 minutes. To this mixture,thionyl chloride (2 L) was added slowly at 30-35° C. over 1-1½ hrs. Themixture was stirred at room temperature for 1-1½ hrs. 50% of thedichloromethane was distilled normally and the remaining 50% was removedby distillation under reduced pressure. The residue was charged withwater (5 L) and ethyl acetate (20 L) and this mixture was stirred for 10minutes.

The ethyl acetate layer was separated and this layer was washed with asoda-ash solution (1 Kg soda-ash in 4 L water) to adjust the pH of thelayer to 8-9. The mixture was stirred for 10 minutes. The ethyl acetatelayer was separated again and was washed with a solution of common salt.(1 Kg NaCl in 3 L water). The mixture was stirred for 10 minutes. Theethyl acetate layer was separated once again and was concentrated underreduced pressure. The residue was taken up with n-hexane (10 L) and wasrefluxed. The product was then cooled to 10° C. and was stirred for 30minutes. The product obtained was filtered through a Nutsche filter andwas washed with n-hexane (1 L). The product,1-(benzyloxy)-4-(chloromethyl)benzene, was dried at 40-42° C. over 5-6hrs. Yield: 75-80% Purity: 90%.

3-(4-(benzyloxy)phenyl)-2-oxopropanal

A 50 L round bottom flask was charged with anhydrous tetrahydrofuran(7.5 L) and magnesium turnings (2.5 Kg) followed by iodine (2 grams) andethyl bromide (10 mL). To this mixture, a solution of1-(benzyloxy)-4-(chloromethyl)benzene (3.2 Kg) in anhydroustetrahydrofuran (40 L) was added drop wise at 40-45° C. The mixture wasstirred for 1 h after which it was cooled to 20° C. To this cooledmixture, a solution of methyl 2,2-dimethoxyacetate (2.5 Kg) in anhydroustetrahydrofuran (2.5 L) was added at 20-38° C. The mixture was stirredfor 30 minutes at 40-42° C. To the Grignard reaction mass, ammoniumchloride solution (2.5 Kg ammonium chloride in 10 L water) was added andthis mixture was stirred for 20 minutes. The tetrahydrofuran layer wasseparated and was concentrated under reduced pressure. The productobtained was purified by column chromatography over silica gel. Thepurified product is heated to 60° C. along with 10 L of 10% HCl for 3hours to obtain 3-(4-(benzyloxy)phenyl)-2-oxopropanal. Yield: 65-70%Purity: 90%.

8-Benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one(coelenterazine)

A 50 L round bottom flask was charged with4-(5-amino-6-benzylpyrazin-2-yl)phenol (0.8 Kg) and3-(4-(benzyloxy)phenyl)-2-oxopropanal (1.4 Kg) followed by 1,4-dioxane(10 L). The reaction mixture was stirred at room temperature for 30minutes. Concentrated hydrochloric acid (1 L) and water (1 L) was addedwhile nitrogen gas was being passed and the reaction mixture was heatedto 80-85° C. for 24 hours in a nitrogen environment. After thecompletion of the reaction, the reaction mixture is cooled to 40° C.then activated carbon (200 g) and activated silica gel (100 g) are addedand filtered.

Meanwhile, another 50 L round bottom flask was charged with HCl (15 L).Add slowly the above reaction mixture in to the HCl at 30-35° C. over1-1½ hrs. The mixture was stirred for 30 minutes at 30-35° C. Theproduct obtained was filtered through a Nutsche filter and was washedwith toluene (1.5 L). The product was dried at 40-45° C. in 8-10 hrs.

The above product (1.5 Kg) was charged with dioxane-HCl (15 L) at roomtemperature. This mixture was stirred for 20 minutes and was then slowlyheated to 60° C. The reaction mixture was stirred at 60-62° C. for 12hrs and it was then stirred for 3 hrs at 70-72° C. The dioxane-HCl wasremoved completely under reduced pressure and the residue was cooled to40° C. The residue was then charged with ethyl acetate (5 L) and themixture was stirred for 15 minutes. The ethyl acetate layer wasdecanted. The residue was charged with dichloromethane (5 L) and thismixture was stirred for 35-40 minutes. Filter the compound through aNutsche filter. To the filtrate dichloromethane (2.5 L) was added andthis mixture was stirred for 25-30 minutes. The compound was filteredonce again and the filtrate material was charged with n-hexane (4 L).The mixture was stirred for 20-25 minutes. The compound,8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one,was filtered once again and was washed with n-hexane (1 L). The product,8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one,was dried under reduced pressure at 40-45° C. over 7-8 hours. Yield: 80%Purity: 60-65%.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the disclosure.

The embodiments of the disclosure in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of makingcoelenterazine, comprising: coupling4-(5-amino-6-benzylpyrazin-2-yl)phenol (coelenteramine) with3-(4-(benzyloxy)phenyl)-2-oxoprop anal to provide8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one; and deprotecting the8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one to provide8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-one (coelenterazine).
 2. The method of claim 1, wherein couplingthe 4-(5-amino-6-benzylpyrazin-2-yl)phenol (coelenteramine) with the3-(4-(benzyloxy)phenyl)-2-oxopropanal is in a solvent mixture comprisingdioxane, water, and HCl.
 3. The method of claim 2, wherein coupling the4-(5-amino-6-benzylpyrazin-2-yl)phenol (coelenteramine) with the3-(4-(benzyloxy)phenyl)-2-oxopropanal is at a temperature of 75° C. to90° C. for 12 to 36 hours in an inert atmosphere.
 4. The method of claim1, wherein deprotecting the8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-onecomprises a first deprotection step of exposing the8-benzyl-2-(4-(benzyloxy)benzyl)-6-(4-hydroxyphenyl)imidazo[1,2-α]pyrazin-3(7H)-oneto HCl in an organic solvent comprising dioxane, and isolating anddrying an intermediate deprotected product.
 5. The method of claim 4,wherein the first deprotection step is at a temperature of 25° C. to 40°C.
 6. The method of claim 4, further comprises a second deprotectionstep of exposing the dried intermediate deprotected product to HCl in anorganic solvent comprising dioxane.
 7. The method of claim 6, whereinthe second deprotection step comprises heating the intermediatedeprotected product in HCl and the organic solvent to a temperature ofabout 70° C. to 75° C. for a duration of 12 to 24 hours to providecoelenterazine.
 8. The method of claim 1, wherein the coelenterazine isobtained in a yield of greater than 70% at a purity of from 55% to 70%relative to 4-(5-amino-6-benzylpyrazin-2-yl)phenol.
 9. The method ofclaim 1, further comprising making the3-(4-(benzyloxy)phenyl)-2-oxopropanal by reacting1-(benzyloxy)-4-(chloromethyl)benzene in two steps to provide3-(4-(benzyloxy)phenyl)-2-oxopropanal.
 10. The method of claim 9,wherein making the 3-(4-(benzyloxy)phenyl)-2-oxopropanal does notinclude more than one palladium-catalyzed reaction.
 11. The method ofclaim 9, comprising a first step of reacting the1-(benzyloxy)-4-(chloromethyl)benzene with methyl 2,2-dimethoxyacetate,ethyl bromide, magnesium, and a catalytic amount of iodine to provide3-(4-(benzyloxy)phenyl)-1,1-dimethoxypropan-2-one.
 12. The method ofclaim 11, wherein the 3-(4-(benzyloxy)phenyl)-1,1-dimethoxypropan-2-oneis purified by silica column chromatography.
 13. The method of claim 12,further comprising a second step of reacting the3-(4-(benzyloxy)phenyl)-1,1-dimethoxypropan-2-one with aqueous HCl toprovide the 3-(4-(benzyloxy)phenyl)-2-oxopropanal.
 14. The method ofclaim 9, wherein the 3-(4-(benzyloxy)phenyl)-2-oxopropanal is isolatedin a yield of 60 to 75% at a purity of 85 to 95% relative to1-(benzyloxy)-4-(chloromethyl)benzene.
 15. A method of making3-(4-(benzyloxy)phenyl)-2-oxopropanal, comprising providing1-(benzyloxy)-4-(chloromethyl)benzene, and reacting the1-(benzyloxy)-4-(chloromethyl)benzene in two steps to provide3-(4-(benzyloxy)phenyl)-2-oxopropanal.
 16. The method of claim 15,wherein the method does not include more than one palladium-catalyzedreaction.
 17. The method of claim 15, comprising a first step ofreacting the 1-(benzyloxy)-4-(chloromethyl)benzene with methyl2,2-dimethoxyacetate, ethyl bromide, magnesium, and a catalytic amountof iodine to provide 3-(4-(benzyloxy)phenyl)-1,1-dimethoxypropan-2-one.18. The method of claim 17, wherein the3-(4-(benzyloxy)phenyl)-1,1-dimethoxypropan-2-one is purified by silicacolumn chromatography.
 19. The method of claim 18, further comprising asecond step of reacting the3-(4-(benzyloxy)phenyl)-1,1-dimethoxypropan-2-one with aqueous HCl toprovide the 3-(4-(benzyloxy)phenyl)-2-oxopropanal.
 20. The method ofclaim 15, wherein the 3-(4-(benzyloxy)phenyl)-2-oxopropanal is isolatedin a yield of 60 to 75% at a purity of 85 to 95% relative to1-(benzyloxy)-4-(chloromethyl)benzene.